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Niemir, Natalia. "Gene transfer in the Sandhoff murine model using a specific recombinant AAV9 vector." Thesis, Paris 5, 2013. http://www.theses.fr/2013PA05S024/document.
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Likhite, Shibi B. "Therapeutic suppression of mutant SOD1 by AAV9-mediated gene therapy approach in Amyotrophic Lateral Sclerosis." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1417394084.
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Kennedy, Zachary C. "Optimizing CRISPR/Cas9 for Gene Silencing of SOD1 in Mouse Models of ALS." eScholarship@UMMS, 2019. https://escholarship.umassmed.edu/gsbs_diss/1047.
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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.
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Stoica, Lorelei I. "Gene Therapy for Amyotrophic Lateral Sclerosis: An AAV Delivered Artifical MicroRNA Against Human SOD1 Increases Survival and Delays Disease Progression of the SOD1G93A Mouse Model: A Dissertation." eScholarship@UMMS, 2015. http://escholarship.umassmed.edu/gsbs_diss/813.
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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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Pacouret, Simon. "Thermostability of Adeno-Associated Virus (AAV) Vectors." Thesis, Nantes, 2018. http://www.theses.fr/2018NANT1041/document.
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Les virus adéno-associés (AAVs) sont des virus à ADN simple brin, nonenveloppés, considérés comme des candidats de choix pour la thérapie génique. Pour augmenter les chances de succès des thérapies géniques basées sur l’AAV, des efforts sont actuellement faits pour développer de nouvelles capsides virales, qui seraient plus résistantes à l’immunité préexistante, plus spécifiques de certains tissus, et compatibles avec une production à grande échelle. L’un des défis posés par le développement de nouveaux vecteurs consiste à comprendre comment conférer de nouvelles fonctions biologiques aux capsides d’AAVs, sans compromettre leur intégrité structurale. Pour ce faire, il est nécessaire d’améliorer notre compréhension des mécanismes gouvernant la métastabilité des capsides d’AAVs. L’objectif de cette thèse était d’étudier la thermostabilité des AAVs, ses liens avec leurs propriétés biologiques, ainsi que ses applications dans le domaine du contrôle qualité des préparations d’AAVs recombinants Dans un premier temps, nous étendons les travaux existants à l’étude de virus AAVs ancestraux (AncAAVs), reconstruits in silico. Nous montrons que Anc80, l’ancêtre commun prédit d’AAV1, 2, 8 et 9, est plus thermostable que ses descendants (ΔT = 15-20°C). Nous identifions ensuite, par une analyse de type phénotype-phylogénie, 12 acides aminés jouant potentiellement un rôle important dans la stabilisation des capsides virales. Nous montrons ensuite que la thermostabilité des capsides d’AAVs, mesurée par fluorimétrie différentielle à balayage (DSF), est utile pour déterminer, à l’échelle protéique, l’identité des préparations de vecteurs viraux, une opération requise par les agences réglementaires. Pour finir, nous appliquons ce test d’identité à l’étude de l’homogénéité structurale des librairies d’AAVs. Ces travaux de thèse pourraient s’avérer utiles pour développement et le manufacturing de nouveaux AAVs recombinants pour la thérapie géniqueAdeno-associated virus (AAV) vectors have emerged as promising gene delivery vehicles for gene therapy. To improve the probability of success of AAV-based therapeutic strategies, efforts are currently being made to engineer novel capsids able to produce and purify well, escape pre-existing immunity, and target specific cell populations more efficiently. One challenge in AAV vector engineering is to understand how to confer new functions to the viral capsid without altering its structural integrity. To do so, there is a critical need to gain further knowledge on the mechanisms steering AAV capsid metastability. The objective of this thesis is to investigate the thermal stability of AAVs, its impact on AAV biology, and applications to quality control of AAV preparations. First, we extend existing thermal stability studies to in silico reconstructed ancestral AAV particles (AncAAVs), and show that, Anc80, the common putative ancestor of AAV1, 2, 8 and 9, is 15-20°C more thermostable than its contemporary homologs. Using phenotype-tophylogeny mapping, we also identify a set of 12 residues potentially playing a key role in capsid metastability. Second, we demonstrate that capsid thermal stability, as measured by Differential Scanning Fluorimetry (DSF), can be used for identification of AAV preparations at the protein level, a requirement of regulatory agencies. Last, we apply this identity assay to the study of capsid mosaic formation in AAV library preparations. This work will help guide the engineering and manufacturing of improved AAV vectors for gene therapy
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Lauramore, Amanda K. "Retinal cell tropism of adeno-associated viral (aav) vector serotypes." [Gainesville, Fla.] : University of Florida, 2004. http://purl.fcla.edu/fcla/etd/UFE0005301.
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Thesis (M.S.)--University of Florida, 2004.Typescript. Title from title page of source document. Document formatted into pages; contains 71 pages. Includes Vita. Includes bibliographical references.
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Crumrine, Jennette Kathleen. "Tissue tropisms of AAV vectors deficient in receptor binding." Connect to resource, 2005. http://hdl.handle.net/1811/440.
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Thesis (Honors)--Ohio State University, 2005.Title from first page of PDF file. Document formattted into pages: contains 40 p.; also includes graphics. Includes bibliographical references (p. 39-40). Available online via Ohio State University's Knowledge Bank.
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Ploquin, Aurélie. "Les vecteurs AAV recombinants : un nouvel outil de vaccination contre les Hénipavirus." Phd thesis, Ecole normale supérieure de lyon - ENS LYON, 2012. http://tel.archives-ouvertes.fr/tel-00756311.
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Les virus Hendra (HeV) et Nipah (NiV) sont des virus émergents appartenant à la famille des Paramyxovirus et au genre des Hénipavirus. Chaque année, ils sont responsables de nombreuses épidémies touchant plusieurs espèces animales dont les hommes, avec une forte morbidité et mortalité. À ce jour, aucun vaccin ni traitement ne sont commercialisés. Ce projet porte sur le développement d'un vaccin génétique pour lutter contre une infection par les Hénipavirus. La stratégie suivie, repose sur l'injection in vivo de vecteurs recombinants dérivés du virus Adéno-Associé (AAVr) codant pour la glycoprotéine d'enveloppe G du virus NiV. Une première expérience réalisée chez la souris, a montré qu'une seule injection de vecteurs AAVr par voie IM permet le développement d'une réponse humorale contre la protéine G, forte et stable dans le temps. Afin de tester le pouvoir protecteur de ce vaccin, des hamsters ont été infectés par les Hénipavirus, compte tenu de leur grande sensibilité à ces infections. L'injection de vecteurs AAVr chez ces animaux a permis de protéger 100 % des animaux infectés par le virus NiV et 50 % des animaux infectés par le virus HeV. Cette étude apporte une nouvelle approche de vaccination et de nouvelles perspectives concernant l'utilisation des vecteurs AAVr pour lutter contre des infections virales émergentes.
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Rouvière, Laura. "Transfert de gènes dans un modèle murin de la maladie de Sandhoff à l'aide d'un vecteur scAAV9 : intérêt d'une double voie d'administration ?" Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCB052/document.
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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
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Khabou, Hanen. "Development of safe and efficient aav vectors for retinal gene therapy." Thesis, Sorbonne université, 2018. http://www.theses.fr/2018SORUS460.
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La vision est notre sens le plus cher et sa perte est un handicap redouté. Or, il existe un ensemble très hétérogène et complexe de dégénérescences rétiniennes héréditaires entraînant une perte de vision irréversible. Aujourd'hui, il n'y a pas de traitement pour ces maladies. Cependant, au cours de la dernière décennie, de nombreuses thérapies géniques ont été testées dans des essais cliniques, donnant de l'espoir pour le traitement des dégénérescences rétiniennes héréditaires. Dans cette thèse, nous avons exploré l'apport des vecteurs viraux dans le contexte général de la thérapie génique rétinienne. Nous avons plus particulièrement optimisé des vecteurs viraux pour des thérapies géniques indépendantes des mutations, largement applicables à toutes les dystrophies rétiniennes avec dégénérescence de bâtonnets puis cônes. Nous avons conçu des vecteurs pour cibler les cônes et étudié leur potentiel de translation pour l'activation optogénétique des cônes dans plusieurs systèmes modèles pertinentsVision is our most cherished sense and its loss is a feared handicap. A highly diverse and complex array of inherited retinal degenerations leads to irreversible vision loss. Today, there is no cure for such disorders. However, in the last decade, many gene therapies entered clinical trials offering hope for the treatment of inherited retinal degenerations. In this thesis, we explored the contribution of viral vectors within the general context of retinal gene therapy. We focused on optimization of viral vectors for mutation-independent gene therapies broadly applicable across rod-cone dystrophies. We carefully designed vectors for targeting cones and studied their translational potential for optogenetic activation of cones in several relevant model systems
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Choudhury, Sourav Roy. "Developing an Adeno-Associated Viral Vector (AAV) Toolbox for CNS Gene Therapy: A Dissertation." eScholarship@UMMS, 2001. http://escholarship.umassmed.edu/gsbs_diss/809.
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Neurological disorders – disorders of the brain, spine and associated nerves – are a leading contributor to global disease burden with a sizable economic cost. Adeno-associated viral (AAV) vectors have emerged as an effective platform for CNS gene therapy and have shown early promise in clinical trials. These trials involve direct infusion into brain parenchyma, an approach that may be suboptimal for treatment of neurodegenerative disorders, which often involve more than a single structure in the CNS. However, overall neuronal transduction efficiency of vectors derived from naturally occurring AAV capsids after systemic administration is relatively low. We have developed novel capsids AAV-AS and AAV-B1 that lead to widespread gene delivery throughout the brain and spinal cord, particularly to neuronal populations. Both transduce the adult mouse brain >10-fold more efficiently than the clinical gold standard AAV9 upon intravascular infusion, with gene transfer to multiple neuronal sub-populations. These vectors are also capable of neuronal transduction in a normal cat. We have demonstrated the efficacy of AAV-AS in the context of Huntington's disease by knocking down huntingtin mRNA 33-50% after a single intravenous injection, which is better than what can be achieved by AAV9 at the particular dose. AAVB1 additionally transduces muscle, beta cells, pulmonary alveoli and retinal vasculature at high efficiency, and has reduced sensitivity to neutralizing antibodies in human sera. Generation of this vector toolbox represents a major step towards gaining genetic access to the entire CNS, and provides a platform to develop new gene therapies for neurodegenerative disorders.
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Choudhury, Sourav Roy. "Developing an Adeno-Associated Viral Vector (AAV) Toolbox for CNS Gene Therapy: A Dissertation." eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/809.
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Neurological disorders – disorders of the brain, spine and associated nerves – are a leading contributor to global disease burden with a sizable economic cost. Adeno-associated viral (AAV) vectors have emerged as an effective platform for CNS gene therapy and have shown early promise in clinical trials. These trials involve direct infusion into brain parenchyma, an approach that may be suboptimal for treatment of neurodegenerative disorders, which often involve more than a single structure in the CNS. However, overall neuronal transduction efficiency of vectors derived from naturally occurring AAV capsids after systemic administration is relatively low. We have developed novel capsids AAV-AS and AAV-B1 that lead to widespread gene delivery throughout the brain and spinal cord, particularly to neuronal populations. Both transduce the adult mouse brain >10-fold more efficiently than the clinical gold standard AAV9 upon intravascular infusion, with gene transfer to multiple neuronal sub-populations. These vectors are also capable of neuronal transduction in a normal cat. We have demonstrated the efficacy of AAV-AS in the context of Huntington's disease by knocking down huntingtin mRNA 33-50% after a single intravenous injection, which is better than what can be achieved by AAV9 at the particular dose. AAVB1 additionally transduces muscle, beta cells, pulmonary alveoli and retinal vasculature at high efficiency, and has reduced sensitivity to neutralizing antibodies in human sera. Generation of this vector toolbox represents a major step towards gaining genetic access to the entire CNS, and provides a platform to develop new gene therapies for neurodegenerative disorders.
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Varadi, Karl [Verfasser], and Jürgen [Akademischer Betreuer] Kleinschmidt. "A novel AAV9 random peptide library to select for endothelial cell – directed gene transfer vectors / Karl Varadi ; Betreuer: Jürgen Kleinschmidt." Heidelberg : Universitätsbibliothek Heidelberg, 2011. http://d-nb.info/1179230132/34.
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Meliani, Amine. "Prevention and inhibition of adverse humoral immune response to gene therapy mediated by adeno-associated virus (AAV) vector." Thesis, Sorbonne université, 2018. http://www.theses.fr/2018SORUS051.
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La thérapie génique peut être définie comme le transfert du gène thérapeutique dans le tissue d’intérêt à l’aide d’un vecteur. A ce jour, les vecteurs dérivés du virus adéno-associés (AAV) représentent les vecteurs de choix pour le transfert de gène in vivo. Cependant, les réponses immunitaires dirigées contre la capside AAV représentent l’obstacle majeur à l’efficacité du transfert de gène médié par le vecteur AAV. Ce travail de thèse a eu pour objectifs de prévenir et d’inhiber la réponse humorale dirigée contre le vecteur AAV. En administrant des nanoparticules contenant de la rapamycine (ou SVP[Rapa]) avec le vecteur AAV, nous avons démontré une inhibition spécifique des réponses humorale et cellulaire dirigées contre la capside AAV. De plus, cette stratégie nous a permis de re-administrer efficacement le vecteur AAV dans des modèles murins et chez le singe. Nos données ont aussi démontré l’élimination des anticorps préexistants dirigés contre le vecteur AAV en administrant SVP[Rapa] avec le bortezomib. Au cours de travail de thèse, nous avons aussi développée et testée l’efficacité des vecteurs AAV associés aux vésicules extracellulaires (vecteurs exo-AAV) à améliorer l’efficacité des vecteurs AAV. En utilisant les vecteurs exo-AAV, nous avons démontré une expression stable et durable du transgène. De plus, les vecteurs exo-AAV ont montré une résistance aux anticorps neutralisants préexistants. Ainsi, au cours de ce projet de thèse des stratégies efficaces ont été développées afin de prévenir et de contrôler les réponses immunitaires dirigés contre le vecteur AAV, permettant ainsi une re-administration efficace du vecteur AAVGene therapy aims to achieve sustained expression of the therapeutic transgene by the introduction of vector cargo into target tissue. To date, viral vectors based on adeno-associated virus (AAV) represent the leading gene delivery tools in vivo. However, immune responses against AAV vector represent the biggest challenge for the widespread use of AAV-based products. In this PhD project, we aimed at the inhibition and prevention of humoral immune responses to AAV capsid. Using nanoparticles containing rapamycin (SVP[Rapa]) given at the time of vector administration, we demonstrated complete abrogation of anti-capsid humoral and cellular immune responses in antigen-specific manner. Using this strategy, we further demonstrated successful vector re-administration in murine models and in non-human primates. Our data also demonstrated elimination of pre-existing antibodies to AAV vector using a combination of SVP[Rapa] and bortezomib. In this PhD thesis project, we also developed and tested the ability of AAV vectors associated with extracellular vesicles (exo-AAV vectors) to enhance AAV vector potency. Using exo-AAV vectors, we demonstrated higher and sustained transgene expression at low vector doses. Exo-AAV vectors also exhibited resistance to neutralization by pre-existing anti-capsid neutralizing antibodies. Thus in this PhD project, powerful strategies have been developed to prevent and control immune responses against AAV vectors, enabling successful AAV vector re-administration
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Golebiowski, Diane L. "Overcoming Toxicity from Transgene Overexpression Through Vector Design in AAV Gene Therapy for GM2 Gangliosidoses." eScholarship@UMMS, 2009. http://escholarship.umassmed.edu/gsbs_diss/895.
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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.
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Golebiowski, Diane L. "Overcoming Toxicity from Transgene Overexpression Through Vector Design in AAV Gene Therapy for GM2 Gangliosidoses." eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/895.
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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.
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Große, Stefanie [Verfasser], and Martin [Akademischer Betreuer] Müller. "Small but increasingly mighty: New insights into Adeno-associated virus (AAV) capsid biology and implications for AAV vector optimization / Stefanie Große ; Betreuer: Martin Müller." Heidelberg : Universitätsbibliothek Heidelberg, 2016. http://d-nb.info/1180735897/34.
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Calero, M. "Novel gene therapy strategies for ADA deficiency using AAV vectors or gene editing." Thesis, University College London (University of London), 2017. http://discovery.ucl.ac.uk/1563615/.
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Rodríguez, Pinhao Miessner Diego. "Production of AAV vectors for gene therapy : a cost-effectiveness and risk assessment." Thesis, Massachusetts Institute of Technology, 2016. http://hdl.handle.net/1721.1/104215.
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Thesis: S.M., Massachusetts Institute of Technology, Department of Chemical Engineering, 2016. In conjunction with the Leaders for Global Operations Program at MIT.Thesis: M.B.A., Massachusetts Institute of Technology, Sloan School of Management, 2016. In conjunction with the Leaders for Global Operations Program at MIT.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 51-56).
Gene therapy is a promising modality for the potential treatment of rare Mendelian diseases. To date a number of high profile proof-of-concept studies within the industry have demonstrated the significant disease-correcting promise of this therapeutic strategy. One of the major hurdles that remains for the commercialization of gene therapies is the lack of efficient manufacturing capabilities for the production of clinical-grade drug substance/drug product. The primary goals for this project were to decrease the biological contamination and cross-contamination risk associated with the biologic manufacturing process for viral gene therapy vectors and to adjust the process in order to optimize commercial profit. The project also included documenting the different existing processes for AAV production and developing a competitive analysis using information from ongoing clinical trials in the industry pipeline. The following process design steps were followed in order to fulfill the project objectives: (1) Define product specifications, analytical needs and market size, (2) Select production platform/process, (3) Collect data and create process flow diagram, (4) Perform material and energy balances, (5) Calculate costs: equipment and consumables, (6) Model the process in a spreadsheet, (7) Carry out sensitivity analyses, (8) Assess cost-effectiveness and risk, and (9) Develop recommendations. Five different AAV production platforms were identified and an AAV gene therapy landscape was generated. Also, the current process that Pfizer is planning to use was documented and an initial market sizing was performed. Finally, all the data necessary to model the process was collected and the cost-effectiveness and biological contamination and cross-contamination risk assessment were completed. This project confirmed that the use of a scalable line of single-use high cell density bioreactors for the production of AAV is cost-effective. This implies that sufficient AAV quantities can be manufactured for preclinical and clinical trials, using the process developed by Pfizer.
by Diego Rodríguez Pinhao Miessner.
S.M.
M.B.A.
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Strobel, Benjamin [Verfasser]. "Modeling pulmonary fibrosis by AAV-mediated TGFβ1 Expression : a proof of concept study for AAV-based disease modeling and riboswitch-controlled vector production / Benjamin Strobel." Konstanz : Bibliothek der Universität Konstanz, 2016. http://d-nb.info/1158496273/34.
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Carty, Nikisha Christine. "Recombinant AAV Gene Therapy and Delivery." Scholar Commons, 2009. https://scholarcommons.usf.edu/etd/1890.
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Alzheimer's disease (AD), first characterized in the early 20th century, is a common form of dementia which can occur as a result of genetic mutations in the genes encoding presenilin 1, presenilin 2, or amyloid precursor protein (APP). These genetic alterations can accelerate the pathological characteristics of AD, including the formation of extracellular neuritic plaques composed of amyloid beta peptides and the formation of intracellular neurofibrillary tangles consisting of hyperphosphorylated tau protein. Ultimately, AD results in gross neuron loss in the brain which is evidenced clinically as a progressive decline in mental capacity. A strong body of scientific evidence has previously demonstrated that the driving factor in the pathogenesis of AD is potentially the accumulation of Aß peptides in the brain. Thus, reduction of Aß deposition is a major therapeutic strategy in the treatment of AD. Recently it has been suggested that Aß accumulation in the brain is modulated, not only by Aß production, but also by its degradation. Several important studies have demonstrated that Aß degradation is modulated by several endogenous zinc metalloproteases shown to have amyloid degrading capabilities. These endogenous proteases include neprilysin (NEP), endothelin converting enzyme (ECE), insulin degrading enzyme (IDE) and matrix metalloprotease 9 (MMP9). In this investigation we study the effects of upregulating expression of several of these proteases through administration of recombinant adeno-associated viral vector (rAAV) containing both endogenous and synthetic genes for ECE and NEP on amyloid deposition in amyloid precursor protein (APP) plus presenilin-1 (PS1) transgenic mice. rAAV administration directly into the brain resulted in increased expression of ECE and NEP and a substantial decrease in amyloid pathology. We were able to significantly increase the area of viral distribution by using novel delivery methods resulting in increased gene expression and distribution.
These data support great potential of gene therapy as a method of treatment for neurological diseases. Optimization of gene transfer methods aimed at a particular cell type and brain region in the CNS can be accomplished using AAV serotype specificity and novel delivery techniques leading to successful gene transduction thus providing a promising therapeutic avenue through which to treat AD.
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Jacques, Steven John. "Evaluation of AAV8 as a gene therapy vector to deliver NT-3 and shRNA_RhoA to injured dorsal root ganglion neurones." Thesis, University of Birmingham, 2012. http://etheses.bham.ac.uk//id/eprint/3461/.
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Two major reasons for the failure of central nervous system axon regeneration are (i) lack of neurotrophic factors available to CNS neurones and (ii) the presence of molecules that inhibit the growth of axons. In this study a gene therapy approach using adeno-associated virus 8 (AAV8) was used to manipulate these two factors. The following major aims were addressed: (i) confirm the bioactivity of transgenes that would be packaged into the AAV8 vector; (ii) assess the cellular tropism of AAV8 in the dorsal root ganglion (DRG); (iii) evaluate the inflammatory responses of the nervous system to AAV8 after intra-DRG and intrathecal injection; (iv) determine the axon regenerative effect of AAV8-mediated delivery of nt-3 (a neurotrophic factor) and shRNA\(_{RhoA}\) (a disinhibitory therapy) to dorsal root ganglion neurones after spinal cord injury in the rat. Delivery of the nt-3 transgene in vitro resulted in production of high levels of NT-3 protein. Transfection of shRNA\(_{RhoA}\)-containing plasmids into cell lines resulted in a marked decrease in the amount of RhoA detectable in cell lysates. AAV8 was found to preferentially transduce large diameter, proprioceptive DRG neurones (DRGN) but in the context of a significant inflammatory response after intra-DRG injection 28d following intra-DRG injection. Axon regenerative effects of AAV8-mediated transgene delivery before lesioning were ambiguous and further work need to be undertaken to clarify this matter.
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Xu, Dan. "Cellular Immunity in Recombinant Adeno-Associated Virus Vector Mediated Gene Therapy." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1313504203.
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Palacios, FaÌbrega JoseÌ Alejandro. "Interaction between the DNA repair machinery and the adeno-associated virus (AAV) : vector transduction and site-specific integration." Thesis, Open University, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.439234.
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Oliveira, Mónica Catarina Castro. "Proteasome-proteins: are these putative targets for basal-like breast cancer therapy with AAV-vectors?" Master's thesis, Universidade de Aveiro, 2016. http://hdl.handle.net/10773/18553.
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Mestrado em Biologia Molecular e CelularO cancro da mama do tipo basal (BLBC) é um grupo de tumores muito agressivo associado a um mau prognóstico. De momento, não existe nenhum tratamento eficaz para o BLBC, uma vez que rapidamente adquirem resistência às terapias normalmente usadas. Assim, é urgente encontrar novas abordagens para tratar esta doença. Com base em dados anteriores, o objetivo geral deste estudo foi avaliar se o PSMA2, uma proteína do proteassoma, seria um alvo putativo para a inibição para terapia em BLBC. Desta forma, o primeiro objetivo específico foi avaliar o efeito anti-tumorigénico de vírus adeno-associados (AAV) capazes de entregar short hairpin RNAs (shRNA), anteriormente validados, capazes de inibir a expressão do PSMA2 em xenotransplantes de células BLBC em ratinho. Para atingir esse objetivo, foram testados in vivo, vetores AAV2 com shRNAs para os genes PLK1 e PSMA2 para diferentes concentrações de partículas virais (2x1010, 2x109, 2x108 partículas virais/tumor), em que células MDA-MB-468 BLBC foram injetadas na mama de ratinhos nude. Após cerca de um mês, foram realizadas injeções intratumorais com AAVs duas vezes por semana. A administração de AAV2-shPSMA2 resultou numa diminuição no crescimento do tumor sem toxicidade evidente, e este efeito foi mais significativo na concentração de 2x109 partículas virais/tumor. O segundo objetivo específico foi analisar a expressão de PSMA2 em amostras humanas de cancro da mama, o que indica que há também uma importância clínica na inibição deste gene, uma vez que se mostrou estar associado a características menos favoráveis relacionadas com tumores da mama do tipo basal. Em conclusão, embora ainda preliminar, os resultados obtidos abrem a possibilidade de direcionar uma terapia genética em BLBC usando vetores AAV recombinantes que entregam shRNAs para silenciar especificamente a expressão do gene PSMA2.
Basal-like breast cancer (BLBC) is an aggressive group of tumours associated to poor patient prognosis. Currently, there is no effective treatment for BLBC once they rapidly acquire resistance to standard therapies. For this reason, novel approaches to treat this disease are urgently needed. Based on previous data, the general goal of this study was to evaluate if PSMA2, a proteasome protein, was a putative target for inhibition in BLBC therapy. In this way, the first specific aim was to evaluate the anti-tumorigenic effect of adeno-associated virus (AAV)-based vectors, that were able to deliver validated short hairpin RNAs (shRNAs) that inhibit the expression of PSMA2 in BLBC mouse xenografts. To achieve that aim, we have tested, in vivo, AAV2 vectors with shRNAs for the genes PLK1 and PSMA2 for different concentrations of viral particles (2x1010, 2x109, 2x108 VP/tumour), MDA-MB-468 BLBC cells were injected into the mammary fat pad of nude mice and, after nearly one month, intratumoral injections with AAVs were performed twice a week. The delivery of AAV2-shPSMA2 resulted in a decrease in tumour growth with no obvious toxicity, and this effect was more significant at the concentration of 2x109 VP/mouse. The second specific aim was to analyse the expression of PSMA2 in human breast cancer samples, which indicated that there is also a clinical importance in inhibiting this gene, once it showed to be associated with less favourable features that are linked to basal-like breast tumours. In conclusion, although still preliminary, the results obtained open a possibility to direct a gene-based therapy in BLBC using recombinant AAVs that deliver shRNAs that specifically silence PSMA2 gene expression.
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Stachler, Matthew D. "Design and engineering of capsid modified AAV-Based vectors targeted towards angiogenic and proliferating vasculature." The Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=osu1180370373.
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Rossi, Axel. "Intracellular fate of AAV particles in human Dendritic Cell and impact on Gene Transfer." Thesis, Lyon, 2016. http://www.theses.fr/2016LYSEN028.
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Les vecteurs viraux dérivés du virus adéno-associé (AAV) apparaissent depuis deux décennies, comme des outils efficaces pour le transfert de gène in vivo. Cependant, malgré une faible immunogénicité et une absence de toxicité in vivo, leur optimisation requiert encore un effort important vers une meilleure compréhension de leur biologie et, en particulier, de leur interaction avec le système immunitaire. Au cours de ce travail de thèse, nous avons utilisé une méthode de sélection dirigée in vitro dans le but d’obtenir un variant de capside capable de transduire efficacement un type cellulaire non-permissif aux vecteurs AAV : les cellules dendritiques (DC). En effet, ces cellules jouent un rôle primordial dans l’établissement de la réponse immunitaire et, par conséquent, dans la persistance de l’expression du transgène in vivo. Cette technologie, très répandue dans la communauté AAV, a permis de sélectionner un variant de capside aux propriétés très intéressantes. La mutation sélectionnée, caractérisée in vitro comme induisant une instabilité de la capside, a permis d’identifier et de surmonter un point de blocage majeur dans le processus de transduction des DC par les vecteurs AAV consistant dans l’étape de décapsidation du génome du vecteur dans le noyau cellulaire. De manière intéressante, le variant obtenu exhibe un avantage en terme de transduction non seulement dans les DC mais aussi dans différents modèles de cellules primaires humaines (e.g. HUVEC) ou animales (OBC), peu ou pas permissive à l’AAV. De plus, des expériences de transfert de gène in vivo réalisées dans un modèle murin, indiquent que le variant sélectionné conduit à une meilleure expression du transgène, possiblement due à la mise en place d’un processus de tolérisation. Les propriétés remarquables de ce variant de capside, font de lui un candidat intéressant pour des applications médicalesVectors derived from the Adeno-associated virus (AAV) have emerged as an efficient system for in vivo gene transfer. However, despite their low immunogenicity and good tolerance in vivo, a better characterization of the host-AAV interaction is required to be able to fully exploit AAV’s potential fora gene therapy or gene vaccination. In this PhD project, we have used an in vitro directed evolution strategy to select an AAV capsid variant able to transduce human dendritic cell (DC), a non-permissive cell type which plays a critical role in the initiation of immune responses and, consequently, on the persistence of the expression of transgene in vivo. This procedure allowed us to identify an AAV variant characterized by a decreased stability of the capsid in vitro. The use of this mutant as a vector to transduce human DC resulted in an improved uncoating of the vector genome in the cell nucleus, thus identifying this step as major barrier toward DC transduction. Interestingly, the selected variant also displayed an increased transduction efficiency not only in DC but also in different primary human and animal cell types, poorly or non-permissive to AAV. Finally, when injected in mice, this AAV variant resulted in a higher expression of the transgene, associated to a low level of immune responses, suggesting the induction of tolerant state. The remarkable features suggest that our selected variant capsid is a promising candidate for medical applications
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Michelfelder, Stefan. "Selection and characterization of targeted vector capsids from random adeno-associated virus type 2 (AAV-2) display peptide libraries." [S.l. : s.n.], 2008. http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-72406.
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Bourdon, Audrey. "Impact de l’inclusion du domaine C-terminal de la dystrophine sur l’efficacité d’une micro-dystrophine dans le cadre d’une thérapie génique de la Dystrophie Musculaire de Duchenne." Thesis, Nantes, 2020. http://www.theses.fr/2020NANT1029.
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La Dystrophie Musculaire de Duchenne (DMD) est une maladie provoquant une dégénérescence progressive de l'ensemble des muscles de l'organisme. Elle est causée par des mutations du gène codant la dystrophine, une protéine indispensable au maintien de l’intégrité des fibres musculaires. Le transfert d’un gène codant une micro-dystrophine (MD) par le biais d’un vecteur recombinant dérivé du virus adéno-associé (AAVr) est l’une des approches thérapeutiques les plus prometteuse pour la DMD. Trois MDs sont actuellement en cours d’évaluation chez des patients. Du fait de la limite d’encapsidation de l’AAVr (5kb), le choix a été fait de ne pas inclure dans ces MDs le domaine C-terminal (CT) de la dystrophine. Il est pourtant connu pour recruter des membres majeurs du complexe protéique associé à la dystrophine (DAPC), qui agit comme médiateur structural et de signalisation au sein des cellules musculaires. Nous avons étudié l’impact de l’inclusion du CT dans une de ces MDs, la MD1, chez le rat DMDmdx, un nouveau modèle animal de la DMD, mimant les pathologies musculaires et cardiaques des patients. Nous avons montré que la MD1 interagit, à des niveaux physiologiques, avec la plupart des partenaires du DAPC dans les muscles squelettiques et cardiaques, et que l'inclusion du CT augmente le recrutement de certains partenaires à des niveaux supra-physiologiques. En parallèle, nous avons démontré que l'inclusion du CT n'améliore pas l'efficacité thérapeutique de la MD1 sur les pathologies musculaires et cardiaques du rat DMDmdx. Nos résultats confirment le potentiel thérapeutique de la MD1 pour la thérapie génique de la DMDDuchenne muscular dystrophy (DMD) is a muscle wasting disorder caused by mutations in the gene encoding dystrophin, a protein essential for the integrity of muscular fibers. Gene therapy using micro-dystrophin (MD) transgenes and recombinant adeno-associated virus (rAAV) vectors is one of the most promising therapeutic approaches for DMD. Three MDs are currently evaluated in patients. Due to the limited packaging capacity of rAAV vectors (5kb), the choice was done to not include dystrophin C-terminal (CT) domain in these MDs. Yet, this domain is known to recruit major members of the dystrophin associated protein complex (DAPC) which act as signaling and structural mediator of muscle cells. We explored the impact of inclusion of the CT in one of these MDs, called MD1, in the DMDmdx rat, a new animal model of DMD, which mimics the clinical pathology at both the muscular and cardiac levels. We showed that MD1 is sufficient to restore interactions at a physiological level of most DAPC partners in skeletal and cardiac muscles, and that inclusion of the CT increases the recruitment of some DAPC partners at supra-physiological levels. In parallel, we demonstrated that inclusion of the CT does not improve MD1 therapeutic efficacy on DMD muscular and cardiac pathologies of DMDmdx rats. Our work strengthens the therapeutic potential of MD1 for gene therapy of DMD
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Pouzolles, Marie. "Reconstitution de l’architecture thymique et de la différenciation des cellules T dans les immunodéficiences génétiques : développement de stratégies thérapeutiques ciblant directement le thymus." Thesis, Montpellier, 2018. http://www.theses.fr/2018MONTT020.
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Les cellules souches hématopoïétiques (CSH) assurent la génération de toutes les lignées sanguines. Leur différenciation en cellules T matures se déroule dans un microenvironnement spécialisé, le thymus, orchestrée par des interactions complexes entre cytokines, chimiokines et cellules stromales. Les mutations bloquant la différenciation des cellules T ont un impact sur l'architecture du thymus, soulignant l’importance des interactions entre cellules T en développement et cellules stromales thymiques.Les déficits immunitaires combinés sévères sont généralement traités, avec succès, par transplantation de CSH allogénique par voie intraveineuse. Cependant, des complications peuvent survenir notamment en cas de greffe non compatible. Pour pallier à cela, la thérapie génique a été développée mais son efficacité et son innocuité restent à améliorer. Dans ce but, notre groupe a développé une approche par correction génique des progéniteurs T directement in vivo, via un vecteur lentiviral. Bien qu’efficace, là encore, l’efficacité de traitement reste insuffisante voire extrêmement limitée chez les macaques.Lors de ma thèse, j'ai donc évalué le potentiel de différents sérotypes de vecteur viraux adéno-associés (AAV) pour la transduction des thymocytes. L'administration IT de plusieurs sérotypes de AAV2 engendre une transduction des thymocytes >10 fois plus élevée que celle des vecteurs lentiviraux. Le sérotype AAV2/8 induit la transduction des thymocytes la plus efficace et les cellules transduites représentent jusqu'à 1% des cellules T périphériques d’une souris immunocompétente. En utilisant des souris immunodéficientes ZAP-70-/- comme paradigme, j'ai découvert que l'injection IT de l’AAV2/8-ZAP-70 entraîne une transduction et différentiation lymphocytaire T rapide, associée à la génération d’une medulla thymique. En effet, des cellules épithéliales thymiques de la médulla (mTEC) exprimant le régulateur auto-immun AIRE sont détectées en <2 semaines. Bien que cette reconstitution soit transitoire, les mTECs AIRE+ diminuant 10 semaines post-injection, les cellules T périphériques corrigées persistent >40 semaines et présentent environ 1 copie du vecteur AAV/cellule. Ces cellules T effectrices peuvent sécréter des niveaux élevés de cytokines et un nombre important de cellules T régulatrices est également généré. Ainsi, une seule vague de thymopoïèse à partir de progéniteurs transduits par l’AAV-ZAP-70, permet une restauration, rapide et transitoire de l'architecture thymique mais, à long terme de cellules T périphériques fonctionnelles.Pour évaluer les diverses populations de TEC régissant le développement et la sélection des cellules T, j'ai collaboré avec les groupes de P Jay/J Abramson/I Amit pour établir une cartographie de novo du compartiment stromal thymique. Nos analyses ont mis en évidence quatre populations majeures de mTEC (I-IV) avec des fonctions distinctes. Notamment, les mTEC-IV constituent une population unique présentant des similarités moléculaires et morphologiques avec les cellules tuft intestinales. Comme nous avions précédemment identifié la sécrétion d'IL-25 par les cellules tufts comme un régulateur des interactions entre compartiment épithélial et hématopoïétique dans l'intestin, nous avons évalué ce potentiel dans le thymus. Ainsi, des souris déficientes en cellules tuft intestinales présentent également une déficience spécifique en mTEC-IV et une homéostasie perturbée de diverses populations exprimant l'IL-25R dans le thymus. Notre recherche a donc permis d'identifier une nouvelle population de TEC tuft avec un rôle critique dans la formation de la niche immunitaire du thymus.L’ensemble de mes résultats montrent le potentiel thérapeutique de stratégie intrathymique de thérapie génique pour des patients ayant besoin d’une reconstitution rapide en cellules T et fournissent de nouvelles perspectives sur les populations stromales thymique et leur rôle dans l’équilibre de la niche immunitaireHematopoietic stem cells (HSC) ensure the generation of all blood lineages. Their differentiation to mature T lymphocytes occurs in the specialized microenvironment of the thymus, orchestrated by complex interactions between cytokines, chemokines, and stromal cells. Mutations resulting in a block in T cell differentiation impact on the architecture of the thymus, pointing to the critical crosstalk between developing T cells and thymic stromal components.Genetic severe combined immunodeficiencies (SCID) are generally treated by the intravenous transplantation of healthy allogeneic HSCs. Although this therapy is often successful, complications can occur, especially for patients receiving non-histocompatible HSC transplants. To circumvent these problems , significant efforts have gone into developing gene therapy strategies but adverse events indicate the necessity of exploring other avenues. Our group hypothesized that in situ gene correction of T lymphoid progenitors in the thymus itself may overcome some of the drawbacks of ex vivo gene therapy. While intrathymic (IT) lentiviral vector administration corrected immunodeficient thymocyte precursors in mice, thymus transduction was inefficient and efficacy in macaques was limited.During my PhD, I assessed the in vivo potential of adeno-associated vectors (AAV) to transduce thymocyte precursors. Intrathymic administration of several different scAAV2 serotypes resulted in a >10-fold higher transduction of thymocytes (3-5%) as compared to lentiviral vectors. scAAV2/8 promoted the highest level of gene transfer and strikingly, transduced cells represented up to 1% of peripheral T lymphocytes in immunocompetent mice. Using ZAP-70-/- immunodeficient mice as a paradigm, I found that IT injection of an AAV2/8-ZAP-70 vector resulted in a rapid transduction and T cell differentiation, correlating with a dramatic generation of the thymus medulla. Indeed, medullary thymic epithelial cells (mTEC) expressing the AIRE autoimmune regulator were detected within <2 weeks. While this reconstitution was transient––AIRE+ mTECs decreased by 10 weeks post gene transfer––gene-corrected peripheral T cells, harboring approximately 1 AAV genome/ cell, persisted for >40 weeks. Effector T cells had the potential to secrete high levels of cytokines and significant numbers of gene-corrected regulatory T cells were also generated. Thus, a single wave of thymopoiesis, from intrathymic AAV-ZAP-70-transduced progenitors, allows for a rapid but transient restoration of the thymic architecture and long-term peripheral T cell function.To better assess the diverse TEC populations that orchestrate T cell development and selection, I collaborated with the groups of P. Jay/J. Abramson/I. Amit to combine single cell analysis and in-vivo fate-mapping to de novo characterize the entire stromal compartment of the thymus. Our analyses highlighted four major medullary TEC (mTEC I-IV) populations with distinct lineage regulator function and specifically, we found that mTEC-IV constitutes a highly divergent TEC subset that bears strong molecular and morphological characteristics to intestinal tuft cells. As we previously identified tuft cell secretion of IL-25 as a regulator of the crosstalk between the epithelial and hematopoietic compartments in the gut, we assessed the potential immune-modulatory function of mTEC-IV. Notably, mice deficient in intestinal tuft cells exhibited a specific depletion of mTEC-IV and a perturbed homeostasis of various IL25-R-expressing populations in the thymus. Taken together, our data identify a new tuft TEC population critical for shaping the thymus immune niche.In conclusion, the data generated during my PhD advance the therapeutic potential of intrathymic-based vector strategies for the treatment of patients requiring a rapid T cell reconstitution and provide new insights into thymic stromal subsets that are critical for shaping the thymus immune niche
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Koo, Taeyoung. "Studies on gene transfer in skeletal muscle cells and tissues using recombinant adeno-associated virus (AAV) vectors." Thesis, Royal Holloway, University of London, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.529039.
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Best, Victoria Maria. "Ongoing cellular responses to transgene products encoded by recombinant adeno-associated virus (rAAV) vectors." The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1262213552.
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Ghenassia, Alexandre. "Induction de réponses mémoires lymphocytaires T CD8 et protection vaccinale après transfert de gènes par le vecteur AAV recombinant." Thesis, Sorbonne Paris Cité, 2015. http://www.theses.fr/2015PA05T032/document.
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La mémoire immunologique est le mécanisme biologique fondamental à la base du développement de la vaccination. La compréhension de ce mécanisme ainsi que de ses interactions avec les différents acteurs du système immunitaire a permis l’élaboration de vaccins qui sont aujourd’hui les garants d’une protection accrue face à l’émergence de maladies infectieuses potentiellement mortelles. La voie d’injection et le mode de transfert de ces vaccins sont des paramètres majeurs à prendre en considération car ils définissent une modulation des réponses immunitaires et de leurs spécificités d’action. De nos jours, seule la voie intramusculaire demeure la voie majoritaire d’administration de vaccins lors de la prophylaxie primaire en santé humaine. Au cours de notre étude, nous nous sommes intéressés à comparer l’injection d’un antigène (l’ovalbumine) selon deux voies d’administration : la voie intramusculaire et la voie intradermique. Nous nous sommes également appuyés sur une technologie du laboratoire qui consiste à transférer des gènes par des vecteurs AAV2/1 recombinants. Nous disposions de deux constructions de ces vecteurs ayant une spécificité pour cibler les cellules musculaires et permettant l’apport d’un effet auxiliaire par les lymphocytes T CD4+ lors d’injections dans des souris femelles. De plus, une de ces constructions nous permettait d’éviter la voie de présentation directe de l’antigène par les cellules dendritiques (DCs) aux lymphocytes T CD8+. Les capacités modulatrices de ces vecteurs nous permirent de montrer pour la première fois que le vecteur AAV2/1 recombinant était capable de faire exprimer un transgène au sein de la peau et d’y générer une réponse cellulaire forte. Nous avons également montré qu’il existait une synergie d’action entre l’effet auxiliaire et la voie intradermique qui améliorait considérablement les réponses cellulaires issues de la présentation croisée d’antigène. Enfin, nous avons pu démontrer que les lymphocytes T CD8+ générés suite à cette synergie d’action présentaient un profil phénotypique de cellules mémoires polyfonctionnelles et capables de protéger l’hôte face à un challenge pathogéniqueImmunological memory is the fundamental biological mechanism at the beginning of the development of vaccination. Understanding this mechanism and its interactions with the various players of the immune system has allowed the development of vaccines that are today the most effective barrier against the emergence of life-threatening infectious diseases. Route of injection and the nature of carriers of these vaccines are key parameters to be taken into consideration because they define a modulation of immune responses and their specific features. Nowadays, only the intramuscular injection route remains the major route of vaccines injection in the context of primary prophylaxis in human health. During our study, we were interested in comparing the injection of antigen (ovalbumin) following two routes of administration: intramuscular and intradermal routes. We also relied on a technology in the laboratory that involves the transfer of genes by rAAV2/1 vectors. We had two constructs of these vectors having specificity to target skeletal muscle cells and allowing us to provide a helper effect from CD4+ T cells during injections into female mice recipients. Moreover, one of these constructs enabled us to avoid the direct presentation of antigens by dendritic cells (DCs) to CD8+ T cells. The capacity of modulation of these vectors allowed us to show for the first time that the rAAV2/1 vector was able to trigger the expression of a transgene in the skin, and there to generate a strong cellular response. We have also shown that CD4+ T cell help and the intradermal route of immunization synergize to improve greatly cellular responses from the cross-presentation of antigens. Finally, we have demonstrated that CD8+ T cells generated following this synergism exhibited a phenotypic profile of polyfunctional memory cells and able to protect the host against a pathogenic challenge
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Steines, Benjamin Richard. "Investigation and application of novel adeno-associated viral vectors for cystic fibrosis gene therapy." Diss., University of Iowa, 2015. https://ir.uiowa.edu/etd/1763.
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Cystic Fibrosis (CF) is a lethal autosomal recessive genetic disorder caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR transports anions at the apical surface of epithelial membranes and functions in many areas of the body. However in CF, loss of CFTR function in the lungs is the major source of morbidity and mortality. Replacing the defective CFTR in the lungs through gene therapy has the potential to cure the disease. Recombinant adeno-associated virus (AAV) is an effective gene transfer vector and has been used extensively to deliver genes to cells in culture. A number of clinical trials using AAV have been attempted for a variety of diseases, including CF, albeit with limited success. Poor vector transduction efficiency prevents effective gene therapy. We have previously used a technique to greatly increase the transduction efficiency of AAV in human lung tissues by selecting from a library of AAVs using a directed evolution technique. However, this evolution was performed in cultured cells and did not fully represent the in vivo environment in which the AAV would be used. In 2008, a CF pig model was developed to develop a further understanding of the mechanisms of CF and CFTR function. We hypothesized that we could use directed evolution to select for a vector in vivo using the pig, allowing gene therapy studies to be conducted in a physiologically relevant model of CF. We selected a novel AAV variant, called AAV2H22, which is closely related to AAV2 but with greatly increased transduction efficiency in pig airway epithelia. AAV2H22 displayed specific tropism for pig airway epithelia and saturated cell surface receptors, indicating specific binding in those cells. We found that AAV2H22-mediated gene transfer corrected chloride and bicarbonate transport defects both in vitro and in vivo. Importantly, bicarbonate transport was sufficient to normalize pH in the airway surface liquid, resulting in increased bacterial killing likely due to increased activity of antimicrobial peptides. To investigate the mechanics of the increased transduction of AAV2H22, capsid mutants were assayed for transduction efficiency. Two of the five amino acid differences between AAV2 and AAV2H22 lie at the surface and are predicted to alter capsid binding. This is consistent with the results showing specific binding in cultured airway epithelia. This research has important implications for gene therapy and investigations using AAV2H22 will increase our understanding of the biology needed to successfully treat CF.
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Kays, Sarah-Katharina [Verfasser], Beatrix [Akademischer Betreuer] Süß, Ulrike A. [Akademischer Betreuer] Nuber, and Christian J. [Akademischer Betreuer] Buchholz. "Receptor-targeted viral vectors: Tracking of stem cells and side by side comparison of AAV and lentiviral vectors / Sarah-Katharina Kays. Betreuer: Beatrix Süß ; Ulrike A. Nuber ; Christian J. Buchholz." Darmstadt : Universitäts- und Landesbibliothek Darmstadt, 2015. http://d-nb.info/1111910707/34.
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Siu, Jason J. Siu. "Hypothalamic Gene Therapy by an Autoregulatory BDNF Vector to Prevent Melanocortin-4-Receptor-Deficient Obesity." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1523884097811049.
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Perdomini, Morgane. "Développement d'une thérapie génique dans le modèle murin cardiaque de l'ataxie de Friedreich en utilisant le vecteur adéno-associé rAAVrh10." Thesis, Strasbourg, 2013. http://www.theses.fr/2013STRAJ105.
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L’ataxie de Friedreich (AF) est une maladie mitochondriale caractérisée par une ataxie spinocérébelleuse et sensitive, une cardiomyopathie et un diabète. L’AF est due à un déficit en frataxine (FXN), une protéine mitochondriale impliquée dans la synthèse des centres Fe-S et l’homéostasie mitochondriale. L’atteinte cardiaque, pour laquelle il n’existe aucun traitement, est la cause principale de décès. Nous avons montré que l’injection intraveineuse d’un vecteur adéno-associé (AAV) rh10 exprimant la FXN humaine prévient le développement de la cardiomyopathie d’un modèle souris de l’AF mais aussi que l’injection du vecteur à des animaux en insuffisance cardiaque permet la correction complète et rapide du phénotype cardiaque. Ces résultats démontrent la capacité des cardiomyocytes défectueux présentant un défaut bioénergétique à être rapidement corrigés. Nous avons ainsi établi la preuve de concept qu’un traitement par thérapie génique est une approche thérapeutique pertinente pour l’AFFriedreich ataxia (FRDA) is a mitochondrial disease with neurodegeneration, hypertrophic cardiomyopathy and diabetes. FRDA is caused by reduced level of frataxine (FXN), an essential mitochondrial protein involved in iron-sulfur cluster biogenesis and mitochondrial homeostasis. Cardiac failure is the most common cause of mortality in FRDA. To date, no treatment exists for FRDA cardiomyopathy. During my PhD, we showed that an adeno-associated vector (AAV) rh10 expressing human FXN injected intravenously not only prevented the onset of the cardiac disease in a faithful FRDA cardiac mouse model, but also, when administered in animals with cardiac failure, reversed rapidly and completely cardiac remodeling and insufficiency. Our results demonstrate the capacity of defective cardiomyocytes with severe energy failure and ultrastructure disorganization to be rapidly corrected and remodeled by gene therapy. Thus, we showed that gene therapy may be a relevant therapeutical approach for FRDA
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Kienle, Eike Christoph [Verfasser], and Hans-Georg [Akademischer Betreuer] Kräusslich. "Secrets to finding the ideal mate: New insights into parameters that govern successful Adeno-associated virus (AAV) vector evolution / Eike Christoph Kienle ; Betreuer: Hans-Georg Kräusslich." Heidelberg : Universitätsbibliothek Heidelberg, 2014. http://d-nb.info/1180300866/34.
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Dupaty, Léa. "Evaluation in vivo de protéines immunorégulatrices dérivées de CTLA-4 et de PD-L1 pour leur capacité à inhiber les réponses immunitaires dans le contexte de la thérapie génique musculaire par AAV." Thesis, Normandie, 2018. http://www.theses.fr/2018NORMR133/document.
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La thérapie génique consiste à introduire du matériel génétique dans des cellules dans l’objectif de traiter une pathologie. Le plus souvent, la thérapie génique s’effectue au moyen d’un vecteur viral, transportant le gène jusque dans les cellules cibles. Dans le cas des maladies monogéniques, l’adeno-associated virus (AAV) s’est imposé progressivement comme un vecteur de choix. Son absence de pathogénicité, son large tropisme et sa capacité à transduire des cellules quiescentes sont autant d’avantages comparés à d’autres vecteurs utilisés en thérapie génique. L’utilisation d’AAV est approuvé en Europe pour le traitement d’un déficit rare en lipoprotéine lipase et vient récemment d’être approuvé par les autorités américaines pour le traitement d’un déficit de la vision. Toutefois, les essais de thérapies géniques se heurtent souvent aux réponses immunitaires dirigées contre l’AAV. En effet, les différents composants de ce vecteur viral ont été identifiés comme pouvant déclencher des réponses immunitaires s’opposant à l’efficacité à long terme de la thérapie génique. De plus, la protéine transgénique peut s’avérer immunogène, ce qui conduit au déclenchement de réponses immunitaires, à la destruction des cellules transduites et in fine à l’échec de la thérapie génique. En clinique, des immunosuppresseurs sont utilisés pour palier à ses effets indésirables. Toutefois, de par leurs effets secondaires infectieux et tumorigènes, des stratégies visant plutôt à induire de la tolérance vis-à-vis de la protéine transgénique, associées à un bénéfice pour la santé des patients, se sont développées.L’objectif de ce travail de thèse a été d’implémenter une nouvelle stratégie visant à étudier l’effet immunorégulateur et tolérogène de protéines de fusion dérivées de CTLA-4/Fc et de PD-L1/Fc. Pour cela, nous avons utilisé un modèle murin récapitulant les réponses immunitaires induites par un AAV permettant l’expression d’une protéine modèle fortement immunogène, l’ovalbumine (Ova). Ensuite, des AAV codant pour les protéines au potentiel immunorégulateur ont été synthétisés et injectés aux souris conjointement à l’AAV-Ova. Cette stratégie d’immunorégulation vectorisée (VIR) nous a permis d’évaluer la capacité de chacune des protéines à moduler les réponses immunitaires dirigées contre l’Ova directement in vivo. Au total, ce travail a permis de mettre en évidence i) l’intérêt et les limites de la stratégie VIR, ii) celui du rôle délétère de CTLA-4/Fc au long terme sur les lymphocytes Tregs CD4+FoxP3+, périphériques et centraux, iii) et de démontrer l’intérêt de 2 nouvelles molécules dérivées PD-L1/Fc sur la persistance de l’OvaGene therapy consist into introducing genetic material into cells to treat genetic disorders. Most gene therapies use viral vectors to carry the gene within target cells. In case of monogenic disorders, adeno-associated viruses (AAV) has become a vector of choice because of its lack of pathogenicity, its large tropism and its capacity to transduce quiescent cells. The use of AAV is approved in Europe to treat a rare lysosomal storage disease and has recently been approved by the FDA to treat a genetic cause of blindness. However, most clinical trials face immune responses directed against AAV components which may be highly immunogenic. This deleterious immunogenicity often lead to the trial failure. In addition, transgenic protein can also be immunogenic, aimaing to the destruction of transduced cells and ultimatly to gene therapy failure. In clinic, immunosuppressive drug remain the only option to counteract unwanted immune responses. These drugs possess infectious and tumorigenic side effects, therefore strategies aiming to rather capable to induce tolerance toward the transgenic protein are being developped and needed. The objectif of this work was to implement a new strategy aiming to study the immunoregulatory and tolerogenic effect of fusion proteins derived from CTLA-4 and PD-L1. We used a murin model recapitulating the immunes responses induced by an AAV coding for an immunogenic model protein, ovalbumin (Ova) presented in previous studies by our group and others. Then, we synthesized AAV coding for our newly designed immunoregulatory protein and injected them into mice along with AAV-Ova. This strategy of vectorized immunoregulation (VIR) allowed to evaluate the intrinsic capacity of each individual proteins to modulate immune responses against Ova directly in vivo. Eventually, this work allow to 1) assess the benefits and limits of the VIR strategy, 2) the deletrious long-term effects of CTLA-4/Fc on central and peripheral Tregs in mice, 3) to demonstrate the interest of new molecules specifically derived from PD-L1/Fc over the immune tolerance through the long-term persistance of Ova transgene
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Baum, Natalie [Verfasser]. "Targeting the EGF-receptor and the CD38/NADase in solid and hematological malignancies with nanobody-based heavy chain antibodies and AAV vectors / Natalie Baum." Hamburg : Staats- und Universitätsbibliothek Hamburg Carl von Ossietzky, 2020. http://d-nb.info/1241743088/34.
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Tshilenge, Kizito tshitoko. "Optimisation du transfert de gène dans les cellules ganglionnaires rétiniennes de chien et de primate non-humain avec un vecteur AAV2 : implications pour le traitement par une approche d’optogénétique du modèle canin RPE65." Thesis, Nantes, 2016. http://www.theses.fr/2016NANT1005/document.
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Les dystrophies rétiniennes héréditaires (DRH) sont un ensemble de pathologies rétiniennes incurable provoquant la cécité. Les DRH sont caractérisées par le dysfonctionnement/dégénérescence des photorécepteurs et le remodelage de la structure de la rétine. Une des approches thérapeutiques envisagées pour traiter les DRH est la thérapie génique spécifique, c’est à dire le remplacement du gène défectueux par un gène sain. Cependant, bien qu’efficace, la thérapie génique spécifique n’est pas toujours applicable, en particulier quand la dégénérescence est trop avancée ou quand le gène muté n’est pas connu. Afin de traiter tous les cas de DRH quelle que soit leur origine génétique et leur stade de progression, une approche de thérapie génique d’addition est envisagée : Le transfert d’optogène. Cela consiste à convertir les cellules encore présentes dans la rétine malgré la dégénérescence, en cellule photosensible suite à l’expression d’un optogène (protéine photosensible). Mon projet de thèse a consisté dans un premier temps à évaluer le transfert de gène avec un vecteur AAV2/2 dans les cellules ganglionnaires rétiniennes de chien et de primate non-humain. Cette première partie a permis d’initier un second projet qui a eu pour objectif d’évaluer l’efficacité du transfert d’optogène (Channelrhodopsin-2) pour la restauration de la fonction visuelle dans un modèle canin de dystrophie rétinienne (le chien Rpe65- /-)Inherited retinal dystrophies (IRD), a group of incurable retinal pathologies, are associated with visual impairments due to a malfunction and/or degeneration of photoreceptors and/or retinal pigment epithelium (RPE). Significant progress in the field of gene therapy has allowed the development and the characterization of an innovative tool to treat IRD patients: recombinant adeno-associated viral vectors (AAV) that carry and deliver therapeutic nucleic acids. However, due to the heterogenic nature of IRD, gene supplementation will not allow to treat all forms of IRD because: (i) the numbers of mutated genes are unknown according to the state of art; (ii) the dominant forms of IRD in which mutations lead to negative effects are not eligible; (iii) the limit of AAV packaging excludes large-sized mutated genes and (iv) this approach is only applicable when photoreceptors are still alive. To treat all IRD patients, a novel therapeutic approach, independently of the mutated gene and the disease kinetic is suitable: the optogene transfer (light-sensitive protein) to restore photosensitivity in neurodegenerative retina by converting surviving retinal cells into photosensors. The primary goal of my research was to promote and characterize adeno-associated virus type 2-(AAV2) transduction in retinal ganglion cells of dog and non-human primate. A second aim was to investigate the feasibility of AAV2-mediated optogenes transfer in retinal ganglion cells as a therapeutic approach to restore visual function in RPE65 deficient dog, a canine model of IRDs
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Sánchez, Daza Anamaría Constanza. "Vectores virales AAV para terapia génica contra el alcoholismo: Inhibición de la enzima aldehído deshidrogenasa mitocondrial en células de hepatoma humanas." Tesis, Universidad de Chile, 2017. http://repositorio.uchile.cl/handle/2250/147444.
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Doctor en Ciencias de la Ingeniería, Mención Ingeniería Química y BiotecnologíaEl alcoholismo es un grave problema socio-económico. El costo asociado al abuso del alcohol se ha estimado en 1.300 millones de dólares para la economía chilena y en 185 billones para Estados Unidos, costo que se debe principalmente a la perdida de la productividad, tratamientos médicos y costos sociales. El etanol es metabolizado en el hígado en dos pasos. El primero depende de la enzima Alcohol deshidrogenasa (ADH) y el segundo de la enzima Aldehído deshidrogenasa (ALDH2). En individuos de la población asiática existe una alta prevalencia de una mutación en el gen de la ALDH2, por lo que tienen una capacidad reducida para metabolizar el acetaldehído, produciendo fuertes efectos como mareos, hipotensión, palpitaciones, etc. Esto resulta en un rechazo al consumo de etanol y protección frente al alcoholismo. Este fenómeno sugiere que la modulación de la expresión de la ALDH2 mediante tecnologías genéticas puede resultar en un fenotipo similar. Por lo tanto, la terapia génica se presenta como una alternativa atractiva para el tratamiento del alcoholismo, simulando el fenotipo asiático, mediante la inhibición específica de la enzima ALDH2, utilizando vectores virales codificantes de un shRNA como herramienta silenciadora. Los virus adeno-asociados (AAV) han sido utilizados como poderosas herramientas para la transferencia de genes en estudios in vivo, en modelos animales y en ensayos clínicos en humanos, con resultados prometedores. La única terapia génica aprobada en el mundo occidental para comercialización es Glybera y está formulada con vectores AAV. En este trabajo se utilizaron vectores scAAV2 que codifican un ALDH2 shRNA, para silenciar la expresión del gen de ALDH2 en líneas celulares humanas. Las líneas celulares HEK-293 y HepG2 se infectaron con el virus scAAV2/shRNA resultando en una reducción de la expresión de ALDH2 a nivel de RNA y proteínas en las dos concentraciones virales probadas (1x104 y 1x105 vg/cel) cada una probada en dos periodos de tiempos. En ambas líneas celulares los niveles de RNA de ALDH2 se redujeron en un 90% y la expresión a nivel proteico se inhibió en un 90% y 52% respectivamente, cinco días después de la infección. Las células HepG2 VL17A (ADH+) tratadas y expuestas a etanol mostraron un aumento de hasta el 50% en los niveles de acetaldehído. Estos resultados sugieren que la terapia génica puede ser una herramienta útil para el tratamiento del alcoholismo, a través del silenciamiento de la expresión de ALDH2 utilizando tecnología shRNA entregada las células por vectores AAV.
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Favre, David. "Evaluation du transfert du gène de l'érythropoi͏̈étine, placé sous le contrôle du système de régulation dépendant de la doxycycline, au moyen d'un vecteur dérivé de l'Adeno-Associated Virus chez le primate." Paris 7, 2002. http://www.theses.fr/2002PA077206.
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Sacristán, Fraile Víctor. "Ingeniería genética del tejido adiposo o del músculo esquelético mediante vectores aav-fgf21 para el tratamiento de la diabetes y la obesidad." Doctoral thesis, Universitat Autònoma de Barcelona, 2018. http://hdl.handle.net/10803/666658.
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La obesidad y la diabetis tipo 2 (DT2) se consideran las dos grandes epidemias del siglo XXI. A pesar del grave problema de salud, económico y social que representan, actualmente no existen terapias completamente efectivas. Asimismo, los tratamientos farmacológicos que se utilizan, frecuentemente presentan importantes efectos secundarios, por lo que es necesario encontrar nuevas aproximaciones terapéuticas para combatir estas epidemias.
Recientemente se ha descrito el factor de crecimiento fibroblástico (FGF21) como un prometedor agente terapéutico tanto para la obesidad como la DT2. FGF21 ejerce su función endocrina en múltiples tejidos diana, regulando la homeostasis energética. Multitud de aproximaciones terapéuticas se han centrado en el desarrollo de péptidos análogos o miméticos que mejoren sus propiedades farmacocinéticas. No obstante, la introducción de modificaciones en la molécula nativa no sólo puede inducir reacción inmunitaria, sino que tampoco evitan su administración periódica. La terapia génica presenta una gran ventaja respecto a estas estrategias terapéuticas, ya que permiten alcanzar niveles circulantes elevados y constantes de la proteína nativa mediante una única administración. Por ello, en esta tesis doctoral se utilizaron vectores virales adenoasociados (AAV) con la finalidad de transducir el músculo esquelético o el tejido adiposo, a fin de mediar elevados niveles circulantes de FGF21 para contrarrestar la obesidad y la DT2.
La administración local de vectores AAV de serotipo 9 codificantes para FGF21 (AAV9-FGF21) en el tejido adiposo epididimal incrementó los niveles circulantes de FGF21 y previno la obesidad y la resistencia a la insulina inducidas por una dieta alta en lípidos en ratones. Asimismo, la administración local de vectores AAV8-FGF21 en el tejido adiposo también fue capaz de revertir la obesidad y la resistencia a la insulina en ratones ob/ob. Los animales tratados con vectores AAV-FGF21 mostraron un incremento del gasto energético y una reducción del depósito de lípidos en el tejido adiposo y en el hígado, así como menor inflamación en los mismos.
La administración local de vectores AAV1-FGF21 en el músculo esquelético de ratones obesos y resistentes a la insulina medió resultados similares a los obtenidos mediante la administración de los vectores AAV8 y AAV9-FGF21 en el tejido adiposo. Además, la sobreexpresión de FGF21 en el músculo esquelético y, el consiguiente aumento de los niveles circulantes de FGF21, previnieron el desarrollo de hepatocarcinomas inducidos por el consumo crónico de una dieta alta en lípidos. Además, la administración de vectores AAV1-FGF21 en el músculo esquelético medó un envejecimiento más saludable de ratones controles, lo que se evidenció por el mantenimiento del peso corporal, una menor adiposidad y niveles de triglicéridos en hígado disminuidos así como una mejora de la resistencia a insulina mediada por la edad.
En conclusión, en esta tesis doctoral se ha demostrado que la ingeniería genética del tejido adiposo y del músculo esquelético mediante vectores AAV codificantes para FGF21 permitió prevenir y revertir la obesidad y la DT2 en modelos murinos obesos y de resistencia a la insulina. Además, el tratamiento con AAV-FGF21 en animales controles promovió que estos animales envejecieran de manera más saludable. Estos resultados proporcionan las bases para la traslación clínica de estas aproximaciones de terapia génica para el tratamiento de la DT2, la obesidad y sus comorbilidades asociadas en humanos en el futuro.Obesity and type 2 diabetes (T2D) are considered the epidemics of the 21st century. Despite the serious health, economic and social problems they represent, no completely effective therapies are available nowadays. Moreover, the current pharmacological treatments display important sides effects. Thus, there is a need to find new therapeutic approaches to combat these epidemics. Fibroblast growth factor 21 (FGF21) is considered a promising therapeutic agent against obesity and T2D. FGF21 exerts its endocrine function on multiple target tissues, regulating energy homeostasis. Many therapeutic approaches have focused on the development of analogue or mimetic peptides with improved pharmacokinetic properties. However, modifications of the native FGF21 can induce an immune reaction and do not avoid periodic administration. Gene therapy has a great advantage over these therapeutic strategies, since it allows reaching high and steady circulating levels of the native protein through a single administration. Therefore, this doctoral thesis used adeno-associated viral vectors (AAV) in order to transduce skeletal muscle or adipose tissue to mediate high circulating levels of FGF21 to counteract obesity and T2D. Local administration of AAV vectors of serotype 9 encoding FGF21 (AAV9-FGF21) in epididymal white adipose tissue increased circulating levels of FGF21 and prevented obesity and insulin resistance induced by a high fat diet (HFD) in mice. Likewise, local administration of AAV8-FGF21 vectors in adipose tissue was also able to reverse obesity and insulin resistance in ob/ob mice. Animals treated with AAV-FGF21 vectors showed increased in energy expenditure and reduction of lipid deposition in adipose tissue and in the liver, as well as lower inflammation in both tissues. Local administration of AAV1-FGF21 vectors in skeletal muscle of obese and insulin-resistant mice mediated similar results to those obtained by administration of AAV8 and AAV9-FGF21 vectors in adipose tissue. In addition, overexpression of FGF21 in skeletal muscle, and the subsequent increase in circulating levels of FGF21, prevented the development of hepatocarcinomas induced by the chronic intake of HFD. Furthermore, the administration of AAV1-FGF21 vectors in skeletal muscle expands healthspan in control mice, what was evidenced by the maintenance of body weight, lower adiposity and triglyceride levels in the liver as well as improved age-realated insulin resistance. In conclusion, the results of this doctoral thesis demonstrated that genetic engineering of adipose tissue and skeletal muscle using AAV vectors coding for FGF21 allowed to prevent and reverse obesity and T2D in murine models of obesity and insulin resistance. In addition, treatment with AAV-FGF21 in control animals improved healthspan. These results provide the basis for the clinical translation of these gene therapy approaches for the treatment of T2D, obesity and their associated comorbidities in humans in the future.
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Ibraheim, Raed R. "Genome Engineering Goes Viral: Repurposing of Adeno-associated Viral Vectors for CRISPR-mediated in Vivo Genome Engineering." eScholarship@UMMS, 2020. https://escholarship.umassmed.edu/gsbs_diss/1114.
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One of the major challenges facing medicine and drug discovery is the large number of genetic diseases caused by inherited mutations leading to a toxic gain-of-function, or loss-of-function of the disease protein. Microbiology offered a new glimpse of hope to address those disorders with the adaptation of the bacterial CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) defense system as a genome editing tool. Cas9 is a unique CRISPR-associated endonuclease protein that can be easily programmed with an RNA [a single-guide RNA (sgRNA)] that is complementary to nearly any DNA locus. Cas9 creates a double-stranded break (DSB) that can be exploited to knock out toxic genes or replenish therapeutic expression levels of essential proteins. In addition to a matching sgRNA sequence, Cas9 requires the presence of a short signature sequence [a protospacer adjacent motif (PAM)] flanking the target locus. Over the past few years, several Cas9-based therapeutic platforms have emerged to correct DNA mutations in a wide range of mammalian cell lines, ex vivo, and in vivo by adapting recombinant adeno-associated virus (rAAV). However, most of the applications of Cas9 in the field have been limited to Streptococcus pyogenes (SpyCas9), which, in its wild-type form, suffers from inaccurate editing at off-target sites. It is also difficult to deliver via an all-in-one (sgRNA+Cas9) rAAV approach due to its large size. In this thesis, I describe other Cas9 nucleases and their development as new AAV-based genome editing platforms for therapeutic editing in vivo in mouse disease models. In the first part of this thesis, I develop the all-in-one AAV strategy to deliver a Neisseria meningitidis Cas9 ortholog (Nme1Cas9) in mice to reduce the level of circulating cholesterol in blood. I also help characterize an enhanced Cas9 from another meningococcus strain (Nme2Cas9) and show that it is effective in performing editing not only in mammalian cell culture, but also in vivo by all-in-one AAV delivery. Additionally, I describe two AAV platforms that enable advanced editing modalities in vivo: 1) segmental DNA deletion by delivering two sgRNAs (along with Nme2Cas9) in one AAV, and 2) precise HDR-based repair by fitting Nme2Cas9, sgRNA and donor DNA within a single AAV capsid. Using these tools, we successfully treat two genetic disorders in mice, underscoring the importance of this powerful duo of AAV and Cas9 in gene therapy to advance novel treatment. Finally, I present preliminary data on how to use these AAV.Nme2Cas9 vectors to treat Alexander Disease, a rare progressive neurological disorder. These findings provide a platform for future application of gene editing in therapeutics.
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Guhasarkar, Dwijit. "A Walk on the Fine Line Between Reward and Risk: AAV-IFNβ Gene Therapy for Glioblastoma: A Dissertation." eScholarship@UMMS, 2007. http://escholarship.umassmed.edu/gsbs_diss/843.
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Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. The current standard-of-care treatment including surgery, radiation and temozolomide (TMZ) chemotherapy does not prolong the survival satisfactorily. Here we have tested the feasibility, efficacy and safety of a potential gene therapy approach using AAV as gene delivery vehicle for treatment of GBM.
Interferon-beta (IFNβ) is a cytokine molecule also having pleiotropic anticancerous properties. Previously it has been shown by our group that AAV mediated local (intracranial) gene delivery of human IFNβ (hIFNβ) could be an effective treatment for non-invasive human glioblastoma (U87) in orthotopic xenograft mouse model.But as one of the major challenges to treat GBM effectively in clinics is its highly invasive property, in the current study we first sought to test the efficacy of our therapeutic model in a highly invasive human GBM (GBM8) xenograft mouse model.
One major limitation of using the xenograft mouse model is that these mice are immune-compromised. Moreover, as IFNβ does not interact with cross-species receptors, the influence of immune systems on GBM remains largely untested. Therefore to test the therapeutic approach in an immune-competent mouse model, we next treated a syngeneic mouse GBM model (GL261) in an immune-competent mouse (C57B6) with the gene encoding the species-matched IFNβ (mIFNβ). We also tested if combination of this IFNβ gene therapy with the current standard chemotherapeutic drug (TMZ) is more effective than any one of the therapeutic modes alone. Finally, we tested the long term safety of the AAV-mIFNβ local gene therapy in healthy C57B6 mice.
Next, we hypothesized that global genetic engineering of brain cells expressing secretory therapeutic protein like hIFNβ could be more beneficial for treatment of invasive, migratory and distal multifocal GBM. We tested this hypothesis using systemic delivery of AAV9 vectors encoding hIFNβ gene for treatment of GBM8 tumor in nude mice.
Using in vivo bioluminescence imaging of tumor associated firefly luciferase activity, long term survival assay and histological analysis of the brains we have shown that local treatment of AAV-hIFNβ for highly invasive human GBM8 is therapeutically beneficial at an early growth phase of tumor. However, systemic delivery route treatment is far superior for treating multifocal distal GBM8 tumors. Nonetheless, for both delivery routes, treatment efficacy is significantly reduced when treated at a later growth phase of the tumor.
In syngeneic GL261 tumor model study, we show that local AAV-mIFNβ gene therapy alone or in combination with TMZ treatment can provide significant survival benefit over control or only TMZ treatment, respectively. However, the animals eventually succumb to the tumor. Safety study in the healthy animals shows significant body weight loss in some treatment groups, whereas one group shows long term survival without any weight loss or any noticeable changes in the external appearances. However, histological analysis indicates marked demyelinating neurotoxic effects upon long term exposures to mIFNβ over-expressions in brain. Overall, we conclude from this study that AAV-IFNβ gene therapy has great therapeutic potential for GBM treatment in future, but the therapeutic window is small and long term continuous expression could have severe deleterious effects on health.
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Guhasarkar, Dwijit. "A Walk on the Fine Line Between Reward and Risk: AAV-IFNβ Gene Therapy for Glioblastoma: A Dissertation." eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/843.
Full textAbstract:
Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. The current standard-of-care treatment including surgery, radiation and temozolomide (TMZ) chemotherapy does not prolong the survival satisfactorily. Here we have tested the feasibility, efficacy and safety of a potential gene therapy approach using AAV as gene delivery vehicle for treatment of GBM.
Interferon-beta (IFNβ) is a cytokine molecule also having pleiotropic anticancerous properties. Previously it has been shown by our group that AAV mediated local (intracranial) gene delivery of human IFNβ (hIFNβ) could be an effective treatment for non-invasive human glioblastoma (U87) in orthotopic xenograft mouse model.But as one of the major challenges to treat GBM effectively in clinics is its highly invasive property, in the current study we first sought to test the efficacy of our therapeutic model in a highly invasive human GBM (GBM8) xenograft mouse model.
One major limitation of using the xenograft mouse model is that these mice are immune-compromised. Moreover, as IFNβ does not interact with cross-species receptors, the influence of immune systems on GBM remains largely untested. Therefore to test the therapeutic approach in an immune-competent mouse model, we next treated a syngeneic mouse GBM model (GL261) in an immune-competent mouse (C57B6) with the gene encoding the species-matched IFNβ (mIFNβ). We also tested if combination of this IFNβ gene therapy with the current standard chemotherapeutic drug (TMZ) is more effective than any one of the therapeutic modes alone. Finally, we tested the long term safety of the AAV-mIFNβ local gene therapy in healthy C57B6 mice.
Next, we hypothesized that global genetic engineering of brain cells expressing secretory therapeutic protein like hIFNβ could be more beneficial for treatment of invasive, migratory and distal multifocal GBM. We tested this hypothesis using systemic delivery of AAV9 vectors encoding hIFNβ gene for treatment of GBM8 tumor in nude mice.
Using in vivo bioluminescence imaging of tumor associated firefly luciferase activity, long term survival assay and histological analysis of the brains we have shown that local treatment of AAV-hIFNβ for highly invasive human GBM8 is therapeutically beneficial at an early growth phase of tumor. However, systemic delivery route treatment is far superior for treating multifocal distal GBM8 tumors. Nonetheless, for both delivery routes, treatment efficacy is significantly reduced when treated at a later growth phase of the tumor.
In syngeneic GL261 tumor model study, we show that local AAV-mIFNβ gene therapy alone or in combination with TMZ treatment can provide significant survival benefit over control or only TMZ treatment, respectively. However, the animals eventually succumb to the tumor. Safety study in the healthy animals shows significant body weight loss in some treatment groups, whereas one group shows long term survival without any weight loss or any noticeable changes in the external appearances. However, histological analysis indicates marked demyelinating neurotoxic effects upon long term exposures to mIFNβ over-expressions in brain. Overall, we conclude from this study that AAV-IFNβ gene therapy has great therapeutic potential for GBM treatment in future, but the therapeutic window is small and long term continuous expression could have severe deleterious effects on health.
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Hardet, Romain. "Inhibition des réponses immunitaires induites après transfert de gène par vecteur AAV recombinant : preuve de concept de l'efficacité prophylactique de la tolérisation orale dans un modèle murin." Rouen, 2016. http://www.theses.fr/2016ROUENR07.
Full textAbstract:
La thérapie génique permet d'envisager de nouvelles solutions thérapeutique dans diverses pathologies telles que les cancers, les maladies monogéniques ou bien encore les infections. Cette approche thérapeutique s'effectue par la transduction de cellules cibles, le plus couramment par le biais de vecteurs viraux. Parmi ces vecteurs, les vecteurs viraux adéno-associés recombinants (rAAV) se sont imposés au fil des études précliniques chez l'animal et des essais cliniques chez l'homme comme un vecteur de choix. Ils offrent en effet de nombreux avantages. Ils sont dérivés de virus non pathogènes, ce qui facilite leur manipulation. Ils sont considérés comme non intégratifs au sein du génome hôte et donc peu susceptibles de promouvoir la tumorogénicité par mutagénèse intentionnelle. Enfin, ils s'avèrent moins immunogènes et moins inflammatoires que d'autres vecteurs viraux tels que les adénovirus. Plus de vingt-cinq ans après le premier essai clinique approuvé chez l'homme, la thérapie génique a connu une histoire tumultueuse, entre les échecs de ses débuts et de grandes avancées menant à des succès thérapeutiques et à la mise sur le marché européen d'un médicament (alipogene tiparvovec). Cependant, l'efficacité de cette approche thérapeutique se heurte encore à de nombreuses limites, qui ne permettent de la qualifier que de prometteuse pour la plupart des patients. Parmi ces limites, l'immunogénicité des vecteurs viraux entrave fortement l'efficacité sur le long terme de la thérapie génique au travers notamment de réponses immunitaires dirigées contre le vecteur et/ou la protéine transgénique. Outre les réponses immunitaires dirigées contre les protéines de la capside, largement documentées, le transgène encapsidé dans le vecteur permet l'expression d'une protéine d'intérêt thérapeutique qui peut elle-même s'avérer immunogène. En effet, en fonction du profil mutationnel à l'origine de la pathologie monogénique, la protéine transgénique peut représenter un néo-antigène et engendrer des réponses immunitaires susceptibles de conduire à l'élimination des cellules transduites. Des traitements immunosuppresseurs systémiques sont utilisés en clinique pour prévenir ces réponses indésirables, mais leur utilisation est connue pour augmenter le risque infectieux et le développement de cancers. Ainsi, des stratégies permettant plutôt d'induire une tolérance immunologique spécifique de la protéine transgénique permettrait d'améliorer l'expression à long terme de la protéine thérapeutique sans fragiliser de manière globale le système immunitaire du patient. L'objectif de ce travail de thèse était d'évaluer dans un modèle murin une telle stratégie reposant sur la tolérisation orale vis-à-vis de la protéine transgénique. La tolérisation orale permet en effet de prévenir l'apparition de réponses immunitaires dirigées contre des antigènes ingérés par voie orale. Dans notre modèle murin d'étude, basé sur l'injection intramusculaire et fortement immunogène d'un vecteur rAAV codant pour l'ovalbumine (Ova), l'administration prophylactique d'Ova diluée dans l'eau de boisson des animaux durant les sept jours précédant ou les dix jours suivant l'injection du vecteur a permis de prévenir totalement les réponses immunitaires dirigées contre la protéine transgénique. L'absence de réponse, aussi bien humorale que cellulaire, a été corrélée à une persistance à long-terme du transgène dans les cellules musculaires transduites ainsi qu'à sa production et sa sécrétion dans le sérum des animaux ainsi traités. L'exploration des mécanismes immunologiques de cette tolérisation a permis de mettre en évidence l'induction d'un état d'anergie des lymphocytes T CD8+ spécifiques de l'Ova durant la phase initiale de tolérisation orale, précédant l'injection du vecteur rAAV-Ova. De manière intéressante, ces cellules sont ensuite délétées du répertoire lymphocytaire suite à l'injection du vecteur rAAV-Ova et à leur réexposition avec l'Ova dans le contexte mimant une thérapie génique musculaire. Ainsi, ce travail a permis d'apporter la preuve de concept de l'efficacité de la tolérisation orale prophylactique vis-à-vis de la protéine transgénique dans le contexte du transfert musculaire de gène par vecteur rAAV. Cette approche pourrait permettre d'envisager, chez certains patients présentant un risque élevé de s'immuniser contre la protéine transgénique, un protocole de tolérisation spécifique de la protéine d'intérêt. Cette stratégie pourrait ainsi améliorer l'expression sur le long terme de la protéine transgénique sans entraver l'ensemble des défenses immunitaires.
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Le, Meur Guylène. "Evaluation d'un vecteur associé à l'adénovirus recombinant de sérotype 4 (AAV4) pour le traitement par thérapie génique d'un modèle canin de l'amaurose congénitale de Leber : le chien Briard RPE65-/-." Nantes, 2007. http://archive.bu.univ-nantes.fr/pollux/show.action?id=343358f5-2fee-48c5-abaf-f33b77afa7f5.
Full textAbstract:
Les vecteurs dérivés des adéno-associated virus (AAV) permettent un transfert de gène efficace et stable dans la rétine. Les futures applications possibles de ce traitement par transfert de gène en ophtalmologie seront le traitement de dégénérescences rétiniennes acquises ou héréditaires. L’Amaurose congénitale de Leber est une forme précoce de dégénérescence rétinienne héréditaire. Un des modèles canins de cette maladie rétinienne est le chien briard RPE65-/-. Après avoir montré que les vecteurs AAV de sérotype 2, 4 et 5 permettaient un transfert stable dans la rétine de chien et de primate non-humain, nous avons évalué un vecteur AAV de sérotype 4, qui cible exclusivement les cellules de l’épithélium pigmentaire, pour le traitement du chien RPE65-/-. Chez les chiots RPE65-/-, ce transfert du gène rpe65 au moyen d’un vecteur AAV4 portant le cDNA humain rpe65 sous le contrôle du promoteur spécifique RPE65, a permis une restauration stable de la fonction rétinienne et de la visionThe vectors derived from the adeno-associated virus (AAV) allow an effective and stable gene transfer in the retina. Possible future applications of this gene therapy in ophthalmology will be the treatment of acquired or hereditary retinal degenerations. Leber congenital amaurosis is an early form of hereditary retinal degeneration. The RPE65-/-briard dog is one of the canine models of this retinal degeneration. After having shown that serotypes 2, 4 and 5 of AAV vectors allowed a stable gene transfer in dog and primate retina, we evaluated an AAV4 vector, which specifically targets the retinal pigmented epithelium, for the treatment of the RPE65-/-briard dog. This rpe65 gene transfer using an AAV4 vector carrying human rpe65 DNA under the control of a specific RPE65 human promoter, allowed a stable restoration of the retinal function and vision in the RPE65-/-treated dogs
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Chtarto, Abdelwahed. "Contribution au développement de nouveaux vecteurs inductibles par la tétracycline et basés sur le parvovirus adéno-associé (AAV)." Doctoral thesis, Universite Libre de Bruxelles, 2005. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210983.
Full textAbstract:
Le parvovirus adéno-associé (AAV) possède un génome à ADN linéaire simple brin de 4,7kb encadré par deux séquences palindromiques inversées et identiques de 145 nucléotides appelées ITRs, requises en cis pour la réplication et l’encapsidation de l’ADN viral. Dans un AAV recombinant (rAAV), la totalité de la partie codante du génome viral est remplacée par une cassette d’expression et seuls les ITRs sont conservés.\
Doctorat en sciences biomédicales
info:eu-repo/semantics/nonPublished