Recombinant adeno-associated virus (rAAV) is a widely used gene therapy vector. Although a wide range of rAAV serotypes can effectively enter most cell types, their transduction efficiencies (i.e., transgene expression) can vary widely depending on the target cell type. Integrins play important roles as co-receptors for rAAV infection, however, it remains unclear how integrin-dependent and -independent mechanisms of rAAV endocytosis influence the efficiency of intracellular virus processing and ultimately transgene expression. In this thesis, I examined the contribution of integrin-mediated endocytosis to transduction of fibroblasts by rAAV2. I found that promoting AAV2/integrin binding with Mn++ greatly enhanced (~17-fold) rAAV2 transduction independently of cell binding and endocytosis. Subcellular localization studies of rAAV2 demonstrated that integrin activation by Mn++ promoted AAV2 aggregation on alpha5 and beta1 integrins and recruitment of the cytosolic integrin effector protein vinculin. Focal adhesion kinase (FAK), a down stream effector of integrin signals, was essential for AAV/integrin complex endocytosis and transduction, but not AAV2 recruitment to integrins. Recruitment of FAK to AAV2/integrin complexes was increased by transiently trapping the endocytic event at the plasma membrane by pharmacologic inhibition of dynasore. This also increased the size of AAV2 clusters found beneath the cell at FAK/integrin complexes resembling immature filopodia and caused a large, FAK-dependent (75-fold) increase in AAV2 transduction. These findings support a model whereby integrin activation at the cell surface can redirect rAAV2 toward a FAK-dependent entry pathway that is more productive for cellular transduction. This pathway appears to be conserved for other rAAV serotypes that contain a capsid integrin-binding domain (AAV1 and 6).

L'obesitat és una malaltia crònica que es caracteritza per una acumulació ectòpica excessiva de greix associada a la inflamació del fetge i del teixit adipós. A més és el principal factor de risc pel desenvolupament de malalties com la diabetis tipus 2 o malalties cardiovasculars. En els darrers temps, degut a la ingesta de dietes enriquides en greixos i al sedentarisme l'obesitat i el sobrepès han adquirit dimensions de pandèmia, fins el punt que la OMS l'ha etiquetat com l'epidèmia del segle XXI on mil milions de persones en tot el món pateixen algun tipus de sobrepès i uns 300 milions de persones es consideren que són obeses com a conseqüència de la sobrenutrició o sobrealimentació permanent en els països industrialitzats.El fetge és l'òrgan responsable de mantenir la homeostasi energètica de tot l'organisme i responsable en gran part del metabolisme de la glucosa i del metabolisme lipídic. A més, el fetge juga un paper fonamental en el desenvolupament de l'obesitat i en la desregulació metabòlica conseqüent. Nombrosos estudis han descrit l'acumulació d'espècies derivades d'àcids grassos en el fetge com a causants dels problemes associats a l'obesitat, ja sigui degut a una aportació excessiva de lípids o a una fallada en l'oxidació. En els individus obesos tot i tenir alts nivells de greix presenten una oxidació d'àcids grassos disminuïda. Per això, en aquesta tesi s'ha estudiat si un augment en l'oxidació d'àcids grassos en el fetge a llarg termini és capaç de reduir l'acumulació lipídica i per tant disminuir l'obesitat ens diferents models d'animals obesos. Per dur a terme aquest objectiu s'ha avaluat l'efecte de la sobreexpressió de la carnitina-palmitoïltransferasa (CPT) 1A, que és l'enzim responsable del control del transport d'àcids grassos en el mitocondri per ser oxidats, mitjançant l'administració de virus adenoassociats (AAVs). Els AAVs són una molt bona eina per utilitzar com a teràpia gènica per la seva capacitat d'expressar els gens que introdueixen durant llargs períodes de temps i per la resposta pràcticament nul·la del sistema immunitari de l'organisme el qual infecten. Els resultats presentats en aquesta tesi demostren que la sobreexpressió de la CPT1A i d'una forma mutada de CPT1A (CPT1AM), insensible al seu inhibidor fisiològic el malonil-CoA, és suficient per augmentar l'oxidació d'àcids grassos en el fetge, reduir l'acumulació lipídica i la inflamació en el fetge i en el teixit adipós. Per tan, la sobreexpressió de la CPT1A i en un major grau la CPT1AM protegeix a l'organisme enfront l'obesitat i els seus problemes associats. Aquests resultats obren un ventall de possibilitats en la recerca de noves teràpies gèniques contra l'obesitat. / Obesity-induced insulin resistance is associated with both ectopic lipid deposition and chronic, low-grade adipose tissue inflammation. Despite their excess fat, obese individuals show lower fatty-acid oxidation (FAO) rates. This has raised the question of whether burning off the excess fat could improve the obese metabolic phenotype. Here, we used human-safe non-immunoreactive adenoassociated viruses (AAV) to mediate long-term hepatic gene transfer of carnitine palmitoyltransferase 1A (CPT1A), the key enzyme in fatty-acid β-oxidation, or its permanently active mutant form CPT1AM, to high-fat diet-treated and genetically obese mice. High-fat diet CPT1A- and, to a greater extent, CPT1AM-expressing mice showed an enhanced hepatic FAO which resulted in increased production of CO2, ATP and ketone bodies. Notably, the increase in hepatic FAO not only reduced liver triacylglyceride content, inflammation and ROS levels but also affected systemically to decrease epididymal adipose tissue weight and inflammation and to improve insulin signaling in liver, adipose tissue and muscle. Obesity-induced weight gain, increase in fasting blood glucose and insulin levels, and augmented expression of gluconeogenic genes were restored to normal only 3 months after AAV treatment. Thus, CPT1A- and, to a greater extent, CPT1AM-expressing mice were protected against obesity-induced weight gain, hepatic steatosis, diabetes and obesity-induced insulin resistance. In addition, genetically obese db/db mice that expressed CPT1AM showed reduced glucose and insulin levels, and liver steatosis. A chronic increase in liver FAO improves the obese metabolic phenotype, which indicates that AAV-mediated CPT1A expression could be a potential molecular therapy for obesity and diabetes.

Adeno-associated viral (AAV) vectors have been shown to be potential vectors for the treatment of diseases, including protocols using RNA interference (RNAi). AAV vector production in insect cells using the baculovirus vector expression system has been a major advance in furthering their use. A major limitation of AAV vector production at high cell densities is a reduction in cell specific yield, which is thought to be caused by nutrient limitations. Nutrient consumption profiles after infection, however, have still not been fully characterized, probably due to the difficulty of characterizing consumption patterns based on increases in cell density, which are minimal after infection. It is known, however, that cells increase in size after infection; therefore, the driving hypothesis of this thesis was that biovolume, or the total volume enclosed by the membrane of viable cells, which accounts for both cell density and cell size, could be used to characterize nutrient consumption patterns both before and after infection. The relationships between nutrient consumption and change in cell density and biovolume were examined by statistical correlation analysis. It was found that in uninfected cultures, no significant correlation differences, using either cell density or biovolume, were observed since cell size remained relatively constant; however, in infected cultures, more than half of the nutrients were found to be better correlated with biovolume than with cell density. When examining the nutrient and metabolite concentration data on a biovolume basis, nutrient consumption remained relatively constant. It is hypothesized that since it has been reported that the rate of cell respiration increases after infection, a more complete oxidation of nutrients occurs to satisfy increased energy needs during infection. By having a basis to base nutrient consumption, we can better assess the needs of the culture. This will allow the development of feeding strategies based on cellular requirements instead of supplying the cultures with generic nutrient cocktails. It is expected that different nutrient mixtures can be used to target different goals such as 1) enhancing cell growth (before infection) and 2) improving the production of recombinant products (after infection). This will not only increase the efficiency of AAV vector production, but will also reduce the cost of production and make the process more economical by eliminating the addition of unnecessary nutrients. Although promising, some limitations of using biovolume still exist. A first limitation is the biovolume measure itself. This measure requires a device that measures cell size, such as a Coulter Counter Multisizer (Beckman-Coulter, Miami, FL, USA), which can be expensive. Capacitance probes can be a more cost effective tool to estimate biovolume; however, the availability of capacitance probes is still not common. A second limitation is the interpretation of the biovolume profiles, which can depend strongly on the fraction of cells in culture that are infected. If the culture is infected asynchronously, then there will be many different cell populations in the culture. Future work may require separating the cell size distribution into populations of viable and non-viable cells to get a better biovolume measure as opposed to assuming that viability is well distributed over the entire range of cell sizes. In infected cultures where the viability may be low, it is likely that the cell size distribution of non-viable cells will be concentrated at the lower end of the distribution (smaller diameter) rather than being well distributed over the whole range. If this is the case, for the infected cultures with low viability, the mean cell diameter calculated will be underestimated, which will lead to an overestimation of nutrient consumption for cultures with low viability. This will certainly affect the accuracy of the nutrient consumption profiles. By separating cell size distribution data into different cell populations of viable and nonviable, the accuracy can be improved.

Adeno-associated viral (AAV) vectors have been shown to be potential vectors for the treatment of diseases, including protocols using RNA interference (RNAi). AAV vector production in insect cells using the baculovirus vector expression system has been a major advance in furthering their use. A major limitation of AAV vector production at high cell densities is a reduction in cell specific yield, which is thought to be caused by nutrient limitations. Nutrient consumption profiles after infection, however, have still not been fully characterized, probably due to the difficulty of characterizing consumption patterns based on increases in cell density, which are minimal after infection. It is known, however, that cells increase in size after infection; therefore, the driving hypothesis of this thesis was that biovolume, or the total volume enclosed by the membrane of viable cells, which accounts for both cell density and cell size, could be used to characterize nutrient consumption patterns both before and after infection. The relationships between nutrient consumption and change in cell density and biovolume were examined by statistical correlation analysis. It was found that in uninfected cultures, no significant correlation differences, using either cell density or biovolume, were observed since cell size remained relatively constant; however, in infected cultures, more than half of the nutrients were found to be better correlated with biovolume than with cell density. When examining the nutrient and metabolite concentration data on a biovolume basis, nutrient consumption remained relatively constant. It is hypothesized that since it has been reported that the rate of cell respiration increases after infection, a more complete oxidation of nutrients occurs to satisfy increased energy needs during infection. By having a basis to base nutrient consumption, we can better assess the needs of the culture. This will allow the development of feeding strategies based on cellular requirements instead of supplying the cultures with generic nutrient cocktails. It is expected that different nutrient mixtures can be used to target different goals such as 1) enhancing cell growth (before infection) and 2) improving the production of recombinant products (after infection). This will not only increase the efficiency of AAV vector production, but will also reduce the cost of production and make the process more economical by eliminating the addition of unnecessary nutrients. Although promising, some limitations of using biovolume still exist. A first limitation is the biovolume measure itself. This measure requires a device that measures cell size, such as a Coulter Counter Multisizer (Beckman-Coulter, Miami, FL, USA), which can be expensive. Capacitance probes can be a more cost effective tool to estimate biovolume; however, the availability of capacitance probes is still not common. A second limitation is the interpretation of the biovolume profiles, which can depend strongly on the fraction of cells in culture that are infected. If the culture is infected asynchronously, then there will be many different cell populations in the culture. Future work may require separating the cell size distribution into populations of viable and non-viable cells to get a better biovolume measure as opposed to assuming that viability is well distributed over the entire range of cell sizes. In infected cultures where the viability may be low, it is likely that the cell size distribution of non-viable cells will be concentrated at the lower end of the distribution (smaller diameter) rather than being well distributed over the whole range. If this is the case, for the infected cultures with low viability, the mean cell diameter calculated will be underestimated, which will lead to an overestimation of nutrient consumption for cultures with low viability. This will certainly affect the accuracy of the nutrient consumption profiles. By separating cell size distribution data into different cell populations of viable and nonviable, the accuracy can be improved.

Age-related macular degeneration (AMD) is the leading cause for visual impairment and blindness in the elder population of developed countries. The primary underlying cause for significant visual loss is the choroidal neovascularization (CNV). Current treatment strategies for AMD include laser photocoagulation, photodynamic therapy (PDT) and excision of neovascular membranes, but have met with limited success. In our previous studies, we demonstrated that gene delivery of angiogenesis inhibitor vasostatin (VS) attenuated the corneal neovascularization in animals. The primary objective of this study was to investigate gene delivery of vasostatin (VS) attenuated the choroidal neovascularization in animals. Retinal and visual function will be evaluated. However, systematic expression of angiogenesis inhibitor may bring adverse effects to physiological processes. The feasibility, efficiency and safety of gene delivery with systemic and local routes were evaluated. Intramuscular polymer-based gene delivery had no side effect such as virus vector and revealed the safety. Recombinant adenovirus (Ad) was used gene delivery system because of its high titer, wide host range, and transduction efficiency. Adeno-associated virus (AAV) represents highly efficient that can facilirate long-term transduction. We propose to improve the efficacy and safety of VS gene delivery, and to search for the effective delivery route and other adjuvant therapy in conjunction with VS for treatment of CNV. Recently, PDT with veteporfin is an established treatment for subfoveal CNV secondary to AMD. We tried to compare the effect and safety of standard and reduced-dose light application PDT in an animal mocel of CNV. The 180-residue VS and its 48-residue (VS48) inhibited the migration and tube formation in cultured endothelial cells. Topical VS application suppresses the progression of laser-induced CNV via angiogenesis ihhibition, as well as in VS48. VS-48 inhibited the growthof vessels in arota rings. Electroretinograms (ERG) analysis revealed that topical VS-48 application for 21 days had no effect on rat retinal functions. Topical VS-48 treatment significantly reversed the CNV-induced alterations in ERG. Transfection of pCMV3-VS into muscle cells resulted in increased production and release of exogenous VS, which specifically inhibited the proliferation of endothelial cells. Rats treated with intrmuscular injection with PVP-VS also showed a significant reduction in the CNV lesions for at least 42 days. Subconjunctival injection with Ad vector revealed no retinal toxicity in ERG. Ad-luciferase via subconjunctival injections showed ocular expression for as long as 112 days by using bioluminescence image analysis in rodent. AAV-luciferase via subconjunctival injections showed ocular expression for as long as 365 days by using bioluminescence image analysis in mice, and AAV serotype 5-luciferase even showed expression lasting for 2 years. Suppression of laser photocoagulation¡Vinduced CNV by Ad-VS was documented in rat model. Combination therapies are important as treatment options. We demonstrated that PDT could effectively attenuate CNV in a rat model, and reduced doses, worked just as well as the standard dose. In the preliminary study of PDT combined topical VS application, treatment led to CNV attenuation more than alone with PDT. The above experiments would enable us to demonstrate that the vasostatin delivery might be a promising strategy for the treatment of AMD and other retinal or ocular disorders. Furthermore, the results from animal studies might be extrapolated for future clinical application.

Rhodopsin-linked retinitis pigmentosa (RP) is the most common form of autosomal dominant RP, an inherited retinal degeneration, in which rod degeneration is followed by secondary cone loss leading to loss of vision and blindness. The overall objective of this work was to develop an optimized gene replacement therapy, delivering the rhodopsin gene for rhodopsin- linked RP and establish whether secondary cone loss can be delayed. A fast-acting single mutant serotype 8 self-complementary adeno-associated virus vector was produced containing the human rhodopsin promoter and the human rhodopsin coding sequence. In vivo studies in rhodopsin knockout mouse showed that the vector administration led to widespread and robust expression of the transgene. Subretinal injection of the vector into three-week pups of rhodopsin knockout mice with cones expressing green fluorescent protein showed restoration of rod-derived electroretinogram (ERG) responses, and preservation of cone- driven ERG responses three months after injection. Similarly, the longitudinal follow-up with confocal scanning ophthalmoscopy found preservation of fluorescent cones up to three months after injection. Overall, these data provided evidence that the designed vector resulted in significant benefit to rod photoreceptors as well as in delay in secondary cone degeneration and built a basis for future use of this vector on dominant models of RP.

Genetically based Central Nervous System (CNS) disorders remain a largely unresolved issue in the world today. Our genome is the source of our greatest strengths and weaknesses. For this reason, intelligent modification of the genome's DNA is a profoundly beneficial goal in the maximization of the overall health of the human race. Potential benefit in this field is currently limited in both effectiveness and safety in regards to the delivery of therapeutic genes into the nucleus, which is protected by many evolution-based barriers. Evolution has also favored the development of highly specialized and infectiously effective viruses capable of overcoming such boundaries. By neutering the naturally occurring and pathologically benign Adeno-Associated Virus (AAV) we have transformed what was once a virus, into a "pure" vector, taking full advantage of evolution's diligent enhancement of these genetic hijackers without introducing unacceptable danger to patients. We utilized the logically engineered, castrated form of AAV serotype 9 (recombinant AAV9/rAAV9) to act as a vehicle for two reporter genes, Enhanced Green Fluorescent Protein (EGFP) and Firefly Luciferase (Fluc) with the goal of assessing and improving the efficiency of vector transduction in murine CNS. We found that rAAV9, when infused into the intrathecal space of mice is capable of extensive and intensive transduction of both neurons and astrocytes throughout the entire length of the SC as well as the hind regions of the brain (brainstem and cerebellum). We also found that efficiency of transduction was best in our highest dose groups, 1E+12 genome copies (GC) in Experiment 1 and 2E+12 GC in Experiment 2, both of which received rAAV9 particles via the two commercially available (100μL and the 200μL) ALZET® Osmotic Pump designs. Administering dosage higher than this directly into the intrathecal space was limited by the size of the pump reservoir and rAAV9 production titer. We are currently attempting to achieve a more complete CNS transduction by performing another experiment in which we place the pump cannula directly into the intracerebroventricular (ICV) space of the lateral ventricles. Our findings reveal that infusion of rAAV9 by intrathecal placed pump cannula is more effective than any other method tested in this study int the transduction of neurons and glial cells of adult&ndashmouse CNS. By elucidating a mode of delivery that maximizes the robustness of transduction efficiency, our results are a critical building block in designing a cure for the array of genetic-based diseases of the CNS, which are currently untreatable

L'activation de la voie β-adrénergique entraîne une augmentation de l'AMPc qui joue un rôle clé dans la régulation de la contraction cardiaque. Alors qu'une stimulation aiguë des récepteurs β-adrénergiques (β-AR) améliore la fonction cardiaque, leur activation chronique dans l'insuffisance cardiaque (IC) est préjudiciable au cœur, car elle favorise la dérégulation du calcium intracellulaire et le remodelage pathologique du cœur. Les phosphodiestérases (PDE) sont responsables de la dégradation de l'AMPc et de la compartimentation, et donc ajustent finement les réponses β-AR. Nous avons montré précédemment que la PDE4B est diminuée dans l'hypertrophie cardiaque pathologique et que l'ablation de PDE4B chez la souris exacerbe la stimulation β-AR du courant Ca2+ de type L et la propension aux arythmies cardiaques. Étant donné qu'un traitement à long terme par des inhibiteurs de la PDE augmente la mortalité dans l'HF, nous avons supposé que la diminution des taux d'AMPc pourrait avoir un effet thérapeutique dans cette maladie. Nous avons exploré si la surexpression cardiaque médiée par les vecteurs viraux adéno-associés sérotype 9 (AAV9) ou à l’aide d’un système transgénique de PDE4B pourrait prévenir une hypertrophie dans un modèle murin d'infusion chronique d'isoprotérénol (Iso) (60 μg / g / jour pendant 2 semaines). L'échocardiographie a permis l'exploration de la fonction cardiaque. L'expression de la protéine PDE4B dans les extraits de coeur a été mesurée par western blot. Des coupes de cœur (10 μm d'épaisseur) ont été prélevées sur des échantillons inclus en paraffine et colorées avec le trichrome de Masson pour quantifier la fibrose. Une augmentation de dix fois et cinq fois des niveaux de protéines PDE4B a été mesurée dans les transgéniques et les AAV9, respectivement. Chez les souris transgéniques adulte, la surexpression constitutive de la PDE4B a provoqué une légère hypertrophie. Chez les souris témoins, de type sauvage ou ayant reçu un AAV9 codant pour la Luciferase(1x1012 particules virales), le traitement par Iso chronique a induit une hypertrophie cardiaque, une fibrose et une diminution de la fraction d'éjection (EF) mesurée par échocardiographie. La surexpression de PDE4B n'a pas empêché l'hypertrophie cardiaque induite par Iso, mais a aboli l'augmentation de la fibrose. Plus important encore, l’EF a été préservé lorsque PDE4B a été surexprimé dans ce modèle pathologique. Au total, ces résultats suggèrent que la thérapie génique avec des AAV9 codant pour PDE est une approche thérapeutique potentielle pour le traitement de l'hypertrophie cardiaque inadaptée. / Activation of the β-adrenergic pathway results in an increase in cAMP which plays a key role in the regulation of cardiac contraction. While an acute stimulation of the β-adrenergic receptors (β-ARs) improves cardiac function, their chronic activation in heart failure (HF) is detrimental to the heart, as it promotes deregulation of intracellular calcium handling and maladaptive remodeling. Multiple phosphodiesterases (PDEs) are responsible for cAMP degradation and compartmentation, and therefore finely tune β-AR responses. We showed previously that PDE4B is decreased in pathological cardiac hypertrophy and PDE4B ablation in mice exacerbates β-AR stimulation of the L-type Ca2+ current and the propensity to cardiac arrhythmias. Since long term treatment with PDE inhibitors increases mortality in HF, we hypothesized that decreasing cAMP levels could have a therapeutic effect in this disease. To address this hypothesis we used two different models: transgenic overexpression of the PDE using the cardiac specific promoter α-MHC, and PDE-encoding adeno-associated virus targeting the heart in adult mice. We explored whether transgenic or serotype 9 adeno-associated viral vectors (AAV9) mediated cardiac overexpression of PDE4B could prevent maladaptive hypertrophy in a mouse model of chronic isoproterenol (Iso) infusion (60 µg/g/day during 2 weeks). Echocardiography allowed cardiac function exploration. PDE4B protein expression in heart extracts was measured by western blot. Heart sections (10 µm thick) were cut from paraffin-embedded specimens and stained with Masson’s trichrome to quantify fibrosis. A ten-fold and five-fold increase in PDE4B protein levels was measured in transgenic and AAV9, respectively. In transgenic mice, constitutive PDE4B overexpression caused a mild hypertrophy in adult mice. In control mice, either wild-type or injected with a AAV9 encoding for (1x1012 vp), chronic Iso treatment induced cardiac hypertrophy, fibrosis, and decreased ejection fraction (EF) measured by echocardiography. Overexpression of PDE4B did not prevent cardiac hypertrophy induced by Iso, but abolished the increase in fibrosis. More importantly, EF was preserved when PDE4B was overexpressed in this pathological model. Altogether, these results suggest that gene therapy with AAV9 encoding PDEs is a potential therapeutic approach for cardiac maladaptive hypertrophy.

Les tauopathies sont des maladies neurodégénératives caractérisées par l’agrégation de protéine Tau. Mis à part ce marqueur pathologique commun, les tauo-pathies présentent une grande diversité, tant sur le plan clinique qu’anatomo-pathologique, qui pourrait résulter de mécanismes pathologiques différents. Un mécanisme commun pourrait impliquer de petits agrégats de type oligomériques comme étant l’espèce toxique dans les tauopathies.L’objectif de ce projet est de déveloper plusieurs modèles de tauopathies, génétiques ou sporadiques, par transfer de gène de la protéine Tau humaine à l’aide de vecteurs adéno-associés. Trois vecteurs sont utilisés, chacun induisant un phénotype spécifique. Tout d’abord, la surexpression de hTAUWT induit une hyperphosphorylation de la protéine associée à une forte mort neuronale, mais en absence d’agrégation.Sa co-expression avec le peptide pro-agrégant TauRD-ΔK280 dans le groupe hTAUProAggr promeut son agrégation, ce qui est associé à des effets neuroprotecteurs. La construction hTAUP301L induit à la fois la formation d’agrégats solubles et matures, associée à un niveau intermédiaire de toxicité. Ces résultats supportent l’idée que les formes solubles oligomériques jouent un rôle crucial dans la pathologie associée à Tau.Ces modèles rapides, obtenus après expression de Tau à des niveaux similaires, récapitulent donc la variabilité phénotypique observée dans les tauopathies humaines. Ils devraient servir dans le futur pour étudier les mécanismes sous-tendant la toxicité des différentes espèces de Tau. Ils pourraient aussi être utiles pour étudier la spécificité et la sélectivité de composés développés pour l’imagerie de Tau par tomographie par émission de positon. / Tauopathies are neurodegenerative diseases characterized by the aggregation of Tau protein. Despite this common hallmark, tauopathies exhibit a wide variety of clinical and anatomo-pathological presentations, which may possibly result from different pathological mechanisms. One hypothesized common mechanism, however, implicates small oligomeric aggregates as drivers of Tau-induced toxicity.The aim of this project was to develop models of sporadic and genetic tauopathies, using adeno-associated viruses to mediate gene transfer of human Tau to the rat brain. Three different constructs were used, each giving rise to a specific phenotype. First, hTAUWT overexpression led to a strong hyperphosphorylation of the protein which was associated with neurotoxicity in absence of any significant aggregation. Its co-expression with the pro-aggregation peptide TauRD-ΔK280 in the hTAUProAggr group strongly promoted its aggregation, with neuroprotective effects. hTAUP301L construct led to aggregation into soluble species as well as mature aggregates accompanied with an intermediate toxicity. Those results support the hypothesis that soluble oligomeric species are key players of Tau-induced neurodegeneration.Those fast developing models, obtained through similar overexpression of human Tau, thus recapitulated the phenotypic variability observed in human tauopathies. Those should prove useful in the future to study mechanisms underlying the toxicity of various Tau species. Those could also serve to study the specificity and selectivity of Tau-directed tracers for positon emission tomography (PET) imaging.

Recombinant adeno-associated virus vectors are one of the most promising gene delivery tools for applications within gene- and cell therapy. The high level of wild-type adeno-associated virus infections in humans is a limitation due to the pre-existing immunity against the vector or its transgene product. An important tool to develop effective and safe therapies is the ability to measure the pre-existing immune responses against the virus capsids in humans. This master thesis at Gyros Protein Technologies aimed to investigate if the Gyrolab immunoassay system can be used to screen for pre-existing anti-capsid immunity in human sera by optimizing and evaluate three different assay formats: an indirect assay, a generic anti-AAV adsorption assay and a bridging assay. The evaluation focused on immunity against adeno-associated virus serotype 2. All immunoassay formats performed well and depending on application, the different formats offers different advantages. The generic anti-AAV adsorption assay offers the ability to easily screen for several viral serotypes without having to label the capsid, and the bridging assay provides high sensitivity. When screening 31 individual human sera, 58% were positive using the indirect assay and the generic anti-AAV adsorption assay and 65% using the bridging assay format. Provided, is automated and high throughout immunoassays where 16 individuals can be screened in one-two hours. It is shown that all three immunoassay formats can be used to screen for anti-adeno-associated virus antibodies, even though further optimization, cut off development and a larger data set is needed to obtain a fully sophisticated screening tool.