Ce travail de thèse a porté sur le développement d'approches thérapeutiques pour les dysferlinopathies, pathologies dues à un déficit en dysferline, pour lesquelle sil n'existe pas de traitement à l'heure actuelle. Nous avons généré et caractérisé un modèle murin déficient en dysferline, B6.A/J-Dysfprmd, pouvant être comparé avec une souche contrôle. Ce modèle présente une atteinte dystrophique dès deux mois et une diminution de l'activité locomotrice est mise en évidence. Dans une approche par transfert de gène dans le muscle, compte-tenu de la taille de l’ADNc de la dysferline, nous avons utilisé la capacité à se concatémériser des vecteurs AAV. Les résultats ont montré une expression stable d’une protéine entière, jusqu’ à au moins un an. Cette expression est associée à une amélioration fonctionnelle et phénotypique des animaux traités. Une autre étude a porté sur l’évaluation du potentiel thérapeutique d’une forme courte de dysferline. Les résultats ont montré qu’une protéine tronquée était exprimée et permet une restauration partielle de la fonction de réparation membranaire, mais aucune amélioration phénotypique n’est détectée. Ces résultats suggèrent pour la première fois, une modularité de la dysferline. Finalement, nous avons testé si un déficit en dysferline pouvait être compensé par la surexpression de myoferline. Un modèle murin surexprimant la myoferline a été créé. Celui-ci ne montre pas de signes délétères dus à cette surexpression. Ces souris transgéniques ont ensuite été croisées avec les souris déficientes en dysferline. Une amélioration de la fonction de réparation membranaire est observée, mais le phénotype dystrophique du muscle n’est pas amélioré. / In this work, I developed and studied therapeutical strategies for dysferlinopathies, neuromuscular disorders due to dysferlin deficiency. To date, no treatment is available for these diseases. We generated and characterized a dysferlin deficient mouse model, B6.A/J-Dysfprmd. The onset begins at 2-month old with an associated decrease of locomotor activity. For gene transfer approach into muscle, dysferlin coding sequence is too large to enter into a single rAAV. We used properties of rAAV to concatemerize by ITR sequences interaction. Results, obtained with this strategy, showed expression of entire dysferline protein until at least one year. We showed also that this expression is associated to an improvement of phenotype and functional activity of tretaed mice. We also studied therapeutical potential of a truncated dysferlin protein. Results showed the expression of a short protein cassociated to a partial functional membrane repair activity, but none in locomotor activity. Nonetheless, these results showed for the first time a partial modularity of dysferlin. Finally, we also studied compensation of dysferlin by myoferlin overexpression in muscle. A myoferlin-overexpressing transgenic mouse model was generated; a significant level of myoferlin is detected but no deleterious effect was observed. These transgenic mice were crossed with dysferlin deficient mice. An improvement of membrane repair capacity is observed, nevertheless, dystrophic features into muscle were still present.

Adeno-associated virus (AAV) is a small, non-pathogenic virus, exploited as a vector for gene therapy applications, with many successful clinical trials. However, these vectors are based on human and non-human primate AAVs, to which there exists pre-existing immunity in the general population. We hypothesized AAVs having low seroprevalence in the human population can be isolated from alternate sources and can be an alternative to those used in current clinical trials. We also hypothesized that the close homology between pig and human tissues suggests that AAVs isolated from pigs would be able to transduce human cells efficiently. Porcine-derived AAVs preferred specific tissue targets when injected in vivo in mice and successfully transduced cells derived from humans. Immune responses generated against the AAV capsid are also important for determining the safety profile of the vectors; there still exists the possibility of the host mounting adverse immune responses against transduced cells, as seen in some of clinical trials. Although the transduction efficiency of AAV gene transfer has been extensively studied in animal models, the host’s immune response towards the gene product is still poorly understood. This thesis addresses the issue by providing a link between protective efficacy against lethal challenge and tissue tropism. Here, AAVs carrying an immunogenic transgene were developed, with the goal to identify those that can protect against lethal challenge of avian flu or Ebola virus in mice, and those that had poor protective efficacy. It was observed that the protective efficacy afforded by an AAV was serotype specific. The protective efficacy and immune responses were compared to the biodistribution and cellular targets of each AAV. Overall, AAVs sharing broad tropism in biodistribution studies had a tendency to protect mice against lethal challenge than those AAVs not found systemically. As well, those AAVs eliciting protective efficacy against lethal challenge were able to transduce antigen-presenting cells including dendritic and B cells. The link between tissue tropism and host immune responses has been poorly understood and this thesis contributes to the AAV field by highlighting the significance of the cellular targets of AAV and this relationship to protective efficacy.

No description available.

La cécité ou la très grande malvoyance peut résulter de différentes pathologies comme les dystrophies rétiniennes héréditaires (DRH) caractérisées par la perte des photorécepteurs. Une des approches pour traiter les DRH est la thérapie génique spécifique, c’est à dire le remplacement du gène défectueux par un gène sain. Des études chez des modèles animaux de DRH ont démontré l’efficacité de la thérapie géniques spécifique, et conduit au lancement d’essais cliniques.Malgré des résultats encourageants, la thérapie génique spécifique n’est pas toujours applicable, en particulier quand la dégénérescence est trop avancée ou si le gène muté est inconnu. Pour traiter tous les cas de DRH, un nouvel axe de thérapie génique est envisagé : le transfert d’optogène.Cette stratégie consiste à réactiver la rétine devenue aveugle par l’expression de protéines photosensibles dans la rétine. Notre objectif est d'évaluer l'efficacité du transfert d'optogène chez un modèle canin naturellement déficient pour le gène Rpe65, provoquant une forme sévère de DRH proche de celles retrouvées chez l’homme. La stratégie thérapeutique retenue est : L'injection intravitréenne, après vitrectomie, d'un vecteur recombinant dérivé du virus adéno-associé de sérotype2 (rAAV2/2), portant le transgène optogénétique sous contrôle d'un promoteur fort et spécifique des tissus neuronaux : hSyn. Le but de ce projet est de transduire efficacement les cellules ganglionnaires rétiniennes d'un modèle canin déficient pour le gène Rpe65 et d'évaluer la photosensibilité des cellules transduites. / Inherited retinal diseases (IRD) affect about 2 million people worldwide, leading to severe visual impairment.Specific gene addition therapy is one of the most promising strategies to treat these patients. Howevermany of them are not eligible for specific gene therapy,such as.1) Patients with unknown deficient genes.2) Patients beyond the therapeutic window.3) Patients whose the deficient gene is too large forAAV encapsidation.4) Patients undergoing a dominant form of IRD.Therefore, the aim of this project is to develop analternative strategy, independent of the mutation and the retinal degeneration kinetic: the optogene transfer. In context of IRD, it will consist to convert survivingretinal ganglion cells into sensitive light cells followingthe transfer of ChR2 or Opn4 optogene. Several rodentmodels of IRD have been successfully treated usingthese optogenes. Nevertheless, this approach hasnever been evaluated in large animal models. The objective of our study will be to define the feasibility ofoptogene transfer to restore vision in blind patients by evaluating the safety and the efficacy of AAV-mediated gene transfer of ChR2, eNpHR or Opn4, after vitrectomy, in ganglion cells of a canine model of IRD, the Rpe65-deficient dog.

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

Gene therapy holds the unprecedented potential to treat disease by manipulating the underlying genetic blueprints of phenotypic behavior. Targeting of gene delivery is essential to achieve specificity for the intended tissue, which is especially critical in cancer gene therapy to avoid destruction of healthy tissue. Adeno-associated virus (AAV) is considered the safest viral vector and, compared to non-viral vectors, offers several advantages: higher efficiency, genetic modification, combinatorial panning, and high monodispersity. Classic viral targeting has focused on engineering ligand-receptor interactions, but many cell surface targets do not support post-binding transduction events. Furthermore, many potential target tissues – such as triple negative breast cancer – may not display a single, unique identifying surface receptor, so new methods of targeting are needed. Alternatively, many pathological states, including most cancers, exhibit upregulation of proteolytic enzymes in the extracellular milieu. The present work describes the development of an AAV platform that has been engineered to activate in response to disease-related proteases. The specificity and sensitivity of these protease-activatable viruses (PAVs) can be tuned to meet the demands of various clinical scenarios, giving the platform some therapeutic versatility. This work represents the first demonstration of a protease-controlled, non-enveloped virus for genetic therapy. These results extend the therapeutic value of AAV, the safest gene vector currently being explored in 73 clinical trials worldwide.

The bestrophinopathies are a set of inherited retinal degenerations caused by mutations in BEST1, and include Best vitelliform macular dystrophy (BVMD), autosomal dominant vitreoretinochoroidopathy (ADVIRC) and autosomal recessive bestrophinopathy (ARB). The corresponding protein, bestrophin-1, is localised to the basolateral membrane of the retinal pigment epithelium (RPE), where it is thought to function as a Ca²⁺-activated Cl- channel. Currently, there are no treatments for these conditions. In recent years, gene therapy has emerged as an exciting treatment option for inherited retinal disorders (IRDs). Gene delivery to retinal cells using a recombinant adeno-associated virus (rAAV) has produced positive results in several IRDs. Given the recessive nature of ARB, this thesis proposes that the rAAV-mediated delivery of bestrophin-1 to the RPE could represent a potential therapy. The aims of this thesis were to produce and compare rAAV vectors in vitro and in vivo for protein expression, localisation following transduction, restoration of chloride conductance in vitro and safety following sub-retinal injection in vivo. Following the production of two rAAV vectors expressing bestrophin-1, western blots confirmed bestrophin-1 protein expression following transduction of HEK293 cells in vitro. Immunocytochemistry (ICC) revealed bestrophin-1 expression that was localised to the cytosol. Whole-cell patch-clamping revealed a significant increase in chloride conductance in HEK293 cells transduced with AAV-BEST1 vectors which was then ablated upon the removal of chloride from the buffers. Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) indicated that the bestrophin-1 protein was successfully transcribed and translated from the BEST1 coding sequence (CDS). Sub-retinal injections of AAV-BEST1 produced bestrophin-1 expression in the RPE of wild-type C57BL/6 mice however significant retinal thinning was seen at higher doses of vector. In conclusion, rAAV-mediated transfer of bestrophin-1 to the RPE has potential to be a future therapy for ARB, however safety issues need to be addressed and an RPE-specific promoter could be more suitable.

No description available.

No description available.

The Adeno-Associated Virus(AAV) is a small, single stranded DNA virus that has been developed as a gene transfer vector. Early clinical trials using recombinant AAV vectors (rAAV) have identified the following concerns that need to be addressed in order to increase efficiency of these vectors. It has since been determined that AAV vector efficiency decreases due to the following mechanisms: ineffective endocytosis, endosomal degradation, inefficient trafficking, and the need to convert from a single-stranded AAV genome to a transcriptional active double-stranded form. The purpose of this study is to elucidate mechanisms that help stabilize the AAV genome in order to make it a more efficient vector for gene therapy. Previously, there have been studies using fluorescent labeling to track the movement of AAV into the nucleus. An integral part of AAV genomic stability may be the obstacles it encounters in the cytoplasm prior to entering the nucleus. Previous studies on improving AAV transduction have focused primarily on the nucleus. The study will hopefully shed light on the hurdles AAV encounters as it moves through the cytoplasm. Thus, this project has designed and utilized a new system for specifically tracking the status of AAV genomes in the cytoplasm as well as in the nucleus. This project utilizes a novel dual luciferase reporter system to track the movement of AAV particles from the cytoplasm into the nucleus to elucidate mechanisms that could contribute to the stabilization of the AAV genome. The novel dual reporter system is comprised of a single-stranded vector containing two different types of secreted luciferases: Cypridina Luciferase and Gaussia Luciferase. Cypridina Luciferase is placed under the control of a nuclear promoter and therefore it is expressed only in the nucleus. The second, Gaussia Luciferase, is under the control of a cytoplasmic promoter that will only be expressed in the cytoplasm upon the presence of T7 RNA polymerase. Using this dual reporter luciferase system along with RT qPCR quantification in a Hek293 cell line expressing the T7 RNA polymerase, demonstrated that genomes are present in the cytoplasm at 18 and 24 hours post infection. The second part of this study is using the dual reporter system to understand the trafficking patterns of rAAV and looking at ways to enhance transduction. One method could be administering rAAV vectors in conjunction with a drug; this approach may help overcome some of these cellular barriers encountered during infection as well as help stabilize the genome. Cidofovir (CDV) is a monophosphate nucleotide analogue that competitively inhibits the incorporation of deoxycytidine triphosphate into viral DNA via viral DNA polymerase. In vitro, CDV actively inhibits a number of DNA viruses including herpes viruses, adenovirus, polyomavirus, papillomavirus, and poxviruses. Cidofovir has already been approved by the Food and Drug Administration (FDA) for the treatment of cytomegalovirus (CMV) retinitis in patients with acquired immunodeficiency syndrome (AIDS). The effects of CDV on small DNA viruses that lack their own viral DNA polymerase, like AAV, has not been documented. Results have demonstrated that CDV is able to increase single-stranded rAAV transgene expression in the nucleus by 2 to 5 fold, depending on the cell type and concentration, in vitro, using both rAAV2 and rAAV8 luciferase reporter vectors. These results have been able to replicated using other reporter vectors: rAAV2-LacZ reporter, rAAV2-GFP, rAAV2-hAAT, and a rAAV8-GFP in vitro. Results have shown a dose-dependent increase in rAAV genomes with CDV pretreatment in HelaS3 cells via Southern Blot. Also southern blot analysis of cells pretreated with CDV then infected with rAAV revealed no difference in the amount of vector present between 0 and 2 hours post infection, suggesting CDV does not enhance viral entry but that CDV may enhance at steps downstream of viral entry. Using RNA sequencing and Ingenuity software analysis of HelaS3 cells pretreated with CDV, an increase in several genes of interest including those involved in the mechanism of viral exit were observed. These genes include Actin and vacuolar proteins, these molecules are involved in and associated with endosomal sorting complexes as well as required for transport inside the cell. This finding along with southern blot data supports the theory that CDV may enhance rAAV trafficking since we observed that CDV pretreatment enhances viral accumulation of rAAV vectors in both the cytoplasm and nucleus 24 hours post-infection. Also utilizing the dual luciferase reporter system an increase in transgene expression present with CDV pretreatment compared to PBS in both the cytoplasm and nucleus was observed suggesting that CDV may enhance with rAAV trafficking. These results taken together demonstrate that this dual reporter system is a powerful tool for understanding and improving rAAV trafficking. Also drugs like CDV can greatly contribute to the understanding of rAAV trafficking, and eventually lead to the development of novel strategies to increase overall efficiency of AAV transduction. / Microbiology and Immunology