Made available in DSpace on 2016-12-23T13:56:08Z (GMT). No. of bitstreams: 1 Dissertacao de Luciana Bueno de Freitas.pdf: 3067234 bytes, checksum: d04ee819c5aa9cdcbded61d436ac75a0 (MD5) Previous issue date: 2009-02-12 / AAV is a parvovirus which depends of a helper virus to develop productive infection. Until now, there are twelve species described, which AAV2, AAV3, AAV5 e AAV9 were described infecting human beings. In the genital tract, the HPV has been the mostly related in association with AAV. A bidirectional interaction between these viruses has been described, which genes of AAV restrain the expression of HPV genes, what could reflect a protector role of AAV on the development of HPV-induced cervical carcinoma. HIV infection seems to increase the persistence and susceptibility to the HPV infection, enhancing the probability to cervical tumors. At the moment, there are no reports about AAV prevalence in patients with HIV infection. This study aimed to investigate the prevalence of AAV and the AAV-HPV coinfection in cervical secretion from HIV seropositive and seronegative women attending at the Reference Center of Sexually Transmitted Disease/AIDS, Vitória-ES. A structured questionnaire was performed to obtain sociodemographic data of all women. Cytological exams were performed and DNA was extracted using the QIAamp® DNA Mini Kit (QIAGEN), as manufacter s instructions. AAV and HPV were investigated by PCR. AAV typing was done by PCR and RFLP. AAV total prevalence was 19.7% (56/284), with 18.7% (21/112) and 20.3% (35/172) in HIV seropositive and seronegative women, respectively. AAV was detected in 27% of HPV-positive women (36/133) and in 13% of the HPV-negative women (20/151), statically significant (p=0.003). The single type detected was AAV2. Cervical findings were: 222 normal cytology, 41 inflammatory alterations, 10 atypical squamous cells of undetermined significance (ASCUS) and 11 cervical intraepithelial neoplasia class 1 and 2 (CIN 1/ 2). The women AAV-HPV-coinfected showed a lesser chance on the development of ASCUS and CIN, compared with those infected only by HPV. The highest prevalence of the AAV2 type is in accordance with other studies, which demonstrate the AAV2 as the most common in human samples. The significant association observed between AAV and HPVinfected women could suggest helper activity of HPV and corroborate previous studies. The presence of AAV reduced the chance of cervical lesions HPV-induced. Moreover, this is the first report concerning AAV prevalence in HIV-infected women and indicates that this infection does not influence the prevalences of AAV or AAV-HPV coinfection / O vírus adeno-associado (AAV) é um parvovírus que depende de vírus helper para que ocorra infecção produtiva. São descritos 12 tipos, sendo que os tipos 2, 3, 5 e 9 infectam seres humanos. No trato genital, o HPV tem sido o vírus helper relatado com maior frequência em associação com o AAV. Interação bidirecional entre estes vírus foi descrita, com o AAV inibindo expressão gênica do HPV, o que pode refletir em um papel protetor do AV no desenvolvimento de carcinoma cervical induzido por HPV. A infecção por HIV aumenta a susceptibilidade à infecção e persistência do HPV, aumentado também a probabilidade de progressão para tumores cervicais. Até o momento, não é conhecida a prevalência de AAV em pacientes infectados por HIV. Este estudo objetivou investigar a prevalência de AAV e de coinfecção AAV-HPV em mulheres soropositivas e soronegativas para HIV atendidas no Centro de Re erência em DST/AIDS, Vitória-ES. Questionário estruturado para obtenção de dados sócio-demográficos foi aplicado a todas as mulheres. Exames citológicos foram realizados nas amostras de espécime cervical e DNA foi extraído pelo kit comercial QIAamp® DNA Mini Kit, seguindo instruções do fabricante. DNA de AAV e HPV foram investigados por PCR. Tipagem para AAV2, 3 e 5 foi realizada por PCR e RFLP. Prevalência total de AAV foi de 19,7% (56/284), sendo 18,7% (21/112) e 20,3% (35/172) em mulheres soropositivas e soronegativas para HIV, respectivamente. O único tipo encontrado de AAV foi o tipo 2. AAV foi detectado em 27% das mulheres positivas para HPV (36/133) e em 13% das negativas para HPV (20/151), sendo significante estatisticamente (p=0,003). Exame citológico revelou: 222 casos normais, 41 com alterações inflamatórias, 10 com ASCUS e 11 com NIC 1 e 2. A chance de desenvolvimento de NIC e ASCUS foi menor nas mulheres coinfectadas por AAV-HPV em relação às infectadas somente por HPV. A detecção de AAV2 nas amostras está de acordo com pesquisas anteriores que demonstram este tipo como o mais frequente em humanos. A significante prevalência do AAV nas mulheres infectadas por HPV sugere atividade helper do HPV e corrobora estudos prévios. Presença do AAV demonstrou reduzir a chance de desenvolvimento de lesões cervicais induzidas por HPV. Este é o primeiro estudo que investigou a prevalência do AAV em mulheres infectadas por HIV e demonstrou que esta infecção não influenciou a prevalência de AAV ou da coinfecção AAV-HPV

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. Le rAAV constitue un outil de choix pour le transfert de gènes dans diverses applications thérapeutiques. Cependant, dans bon nombre d’entre elles, il est nécessaire de pouvoir moduler l’expression du transgène quantitativement et au cours du temps. Plusieurs systèmes de régulation ont été décrits dont le système d’activation (Tet-On) de l’expression du transgène par la tétracycline et ses analogues (ex : la doxycycline). Le transfert et l’activation de l’expression du transgène par la doxycycline (Dox) nécessite deux vecteurs d’expression, un premier vecteur dans lequel le transactivateur (rtTA) est exprimé à partir d’un promoteur constitutif et un second qui porte le gène d’intérêt sous le contrôle du promoteur tétracycline (Ptet). Le Ptet est constitué du promoteur minimal du cytomégalovirus humain (PhCMVmini) placé en aval d’une répétition de séquences dites "opérateurs" (tetO). En présence de la Dox, le rtTA change de conformation, se lie au tetO et active la transcription du gène d’intérêt à partir du PhCMVmini. Pour le transfert de gène in vivo, il est cependant préférable de disposer d’un vecteur portant les deux cassettes d’expression au sein d’une seule construction (rAAV unique). Toutefois, les ITRs d’AAV d’une part et les séquences "enhancers" du promoteur utilisé pour exprimer le rtTA d’autre part, interfèrent avec le Ptet donnant lieu à une expression du gène d’intérêt à l’état non induit et par conséquent à un faible facteur d’induction. Nous décrivons dans ce travail un vecteur rAAV unique dont l’expression du transgène est activée par la tétracycline après transfert dans le cerveau de rat. En effet, nous avons développé un vecteur autorégulable dans lequel les deux cassettes d’expression sont placées en orientations opposées et la transcription du transgène et du rtTA est initiée à partir d’un promoteur tétracycline bidirectionnel (Ptet-bidi) et terminée par les signaux de polyadénylation bidirectionnels de SV40. Placées à côté de chaque ITR, ces dernières séquences pourraient également servir à arrêter la trancription à partir des ITRs d’AAV en absence de l’inducteur. Les performances de notre vecteur portant le gène rapporteur egfp (rAAV-ptetbidi-EGFP) ont été établies dans diverses lignées cellulaires immortalisées, dans des cultures primaires de cellules de Schwann ainsi que dans le cerveau du rat et des facteurs d’induction allant de 20 à 100 fois ont été observés. Nous avons également évalué la capacité de la minocycline (Mino), un antibiotique de la famille des tétracyclines utilisé pour ses propriétés anti-inflammatoires dans le cerveau, à induire l’expression du transgène à partir du Ptet dans une lignée de cellules U87-MG exprimant de façon stable le plasmide ptetbidi-EGFP. Quoique l’induction maximale de l’expression du transgène par la Mino nécessite des doses plus élevées et un temps plus long de traitement comparée à la Dox, elle apparaît moins toxique à des doses effectrices. Nous avons également évalué la réversibilité du système. Les résultats montrent une extinction plus rapide dans des cellules induites par la Mino comparée à celle obtenue dans des cellules induites par la Dox. Cependant, la cinétique d’induction du rAAV-ptetbidi-EGFP était lente et le niveau basal d’expression était encore élevé. De plus, à l’état induit, le nombre de cellules transduites par ce vecteur in vitro et in vivo reste inférieur à celui obtenu avec un vecteur équivalent portant le transgène sous le contrôle d’un promoteur constitutif. Nous avons réussi à améliorer l’inductibilité de notre vecteur portant le gène rapporteur egfp ou le gène thérapeutique hgdnf codant pour un facteur neurotrophique ayant un effet neuroprotecteur sur les neurones dopaminergiques mais également des effets non désirés : i) en plaçant, en aval du rtTA, le WPRE, une séquence de régulation post-transcriptionnelle d’origine virale permettant l’accumulation du transactivateur à concentration plus élevée dans les cellules transduites. Il en résulte un démarrage plus rapide et un niveau plus élevé de l’expression du transgène ainsi qu’une augmentation du nombre de cellules transduites dans le striatum de rat en réponse à la Dox; ii) en remplaçant le rtTA par le rtTA2SM2 moins toxique, plus stable et ayant une meilleure affinité de liaison au tetO. L’utilisation du rtTA2SM2 permet une réduction du niveau basal d’expression du transgène et son induction à plus faible dose d’inducteur. La version améliorée de notre vecteur a été ensuite encapsidée dans le sérotype 1 d’AAV, qui, injecté dans le striatum de rat, permet d’améliorer le volume de transduction et d’augmenter le nombre de cellules "GFP-positives" transduites comparé au sérotype 2 couramment utilisé. Un facteur d’induction de l’ordre de 10 fois a été également obtenu au moyen d’un rAAV1-ptet-bidi-hGDNF avec une quantité de GDNF exprimée à l’état induit dans la gamme des concentrations neuroprotectrices (100 pg/mg de tissu).

<p>Glycogen storage disease type Ia is an autosomal recessive disorder caused by a mutation in the glucose-6-phosphatase (G6Pase) catalytic subunit, encoded in humans by G6PC. G6Pase dephosphorylates glucose-6-phosphate (G6P) in the liver to generate glucose that can be shuttled to the bloodstream to maintain normoglycemia. Patients with GSD Ia typically present at 6 months of age with sever hypoglycemia, which is lethal if untreated. The current treatment is a strict dietary regimen in which children must be fed every 2 hours overnight or given nasogastric tube feeding, and adults must consume uncooked cornstarch around the clock to maintain normal blood sugar levels. This treatment maintains survival but fails to prevent other symptoms related to metabolism of the excess G6P, and patients develop hepatic adenomas that may become hepatocellular carcinoma later in life, in addition to progressive renal complications.</p><p>To overcome the problems persisting during dietary therapy, the Koeberl lab has sought to develop gene therapy approaches that use adeno-associated virus (AAV) vectors to replace the G6pase activity, restoring normoglycemia and normal metabolic processes. However, the vast majority of AAV-delivered genetic material exists as episomes that do not replicate as cells divide, so the effects of AAV gene therapy on GSD Ia mouse and dog models have proven temporary. We hypothesized that driving integration of therapeutic vector genomes into an affected individual's genome would improve beneficial effects' longevity.</p><p>We tested several approaches to accomplish this, and have found positive effects using a zinc finger nuclease (ZFN) that targets the mouse safe harbor ROSA26 locus to induce homologous recombination of the G6PC donor vector into the mouse genome. We were able to see an improvement in mouse survival to 8 months of age, an increase in G6Pase activity at 3 months of age, and a decrease in glycogen accumulation at 3 months of age, when the ZFN vector is administered alongside the G6PC vector, compared with mice that received the G6PC vector alone.</p><p>We have also taken an alternative approach to overcoming the long-term complications of the current dietary treatment, which would augment rather than replace the current treatment. We have examined several drugs known to induce autophagy in other disease models or cell culture systems, to determine if we could manipulate autophagic activity in G6PC knockdown hepatocytes or GSD Ia mice. We have found positive results using rapamycin, a well-studied MTOR inhibitor, in mice and cells, and have screened several other drugs as well, finding positive effects for bezafibrate, mifepristone, carbamazepin, and lithium chloride, in terms of lipid reduction (which accumulates as a symptom of GSD Ia) and/or LC3-II enhancement, which is reduced in GSD Ia due to downregulation of autophagy during G6P accumulation.</p> / Dissertation

Adeno-associated Virus (AAV) Rep proteins are multifunctional proteins that carry out various DNA transactions required for the life cycle of AAV. The Rep proteins have been found to be important for genome replication, gene regulation, site-specific integration and play an essential role in genome packaging. There are two main groups of Rep proteins: large and small Reps; both groups are SF3 helicase family members. During DNA packaging, studies have shown that the small Rep proteins are critical to produce fully packed particles. Using stopped-flow kinetic analysis, we show a significant difference in helicase activity between the small and large Rep proteins that support the notion that the small Rep proteins are the primary motor to package DNA due to more efficient motor activity. That leaves the large Rep proteins to serve a different role during packaging. In previous studies, we have shown that the large Rep proteins have the ability to change their oligomeric state depending on the nature of the DNA substrate. We can observe double octameric rings with single-stranded DNA (ssDNA) and heptameric complex with double-stranded DNA (dsDNA). To understand Rep protein structural plasticity, we solved a 6.96 Å cryo-EM structure of Rep68*/ssDNA complex illustrating that the formation of Rep octamer rings is dominated by interactions between their N-terminal origin-binding domain (OBD) using the same interface utilized to recognize dsDNA specifically. Our analysis of the structural data suggests that the double octameric ring structure is stabilized by ssDNA that bridges octameric rings together. The structure shows that the helicase domains are highly flexible and that ssDNA is present at the center of the ring. In addition, we have solved a preliminary 12 Å model of Rep68*/dsDNA complex showing a heptameric ring encircling a DNA molecule. Our structural and functional data offer insights to the various Rep-DNA scaffolds that can perform diverse functions during the AAV life cycle.

Cystic fibrosis (CF) is a lethal autosomal recessive genetic disease caused by mutations in a single gene, the cystic fibrosis transmembrane conductance regulator (CFTR). CF affects multiple organ systems, but the major cause of morbidity and mortality is due to disease in the lungs. In theory, using gene therapy to deliver a correct copy of CFTR to the cells of the airway epithelium could result in a lifelong cure. Adeno-associated virus (AAV) is a single stranded DNA virus that is a promising candidate vector for gene therapy of multiple diseases, and numerous clinical trials are currently underway. Despite recent clinical successes, several challenges still impede wider application of AAV gene therapy to numerous diseases, including CF, as AAV-mediated gene transfer to the airways remains below the level needed for therapeutic efficacy for CF. We hypothesized that the low transduction efficiency of AAV in the airways could be overcome by using directed evolution of AAV in organotypic human and pig airway models, and in vivo in the lungs of pigs to select novel AAV capsid variants with improved infectious properties. We discovered a highly infectious, novel AAV that was a chimera of AAV2 and AAV5 with one point mutation (A581T) which we called AAV2.5T. We found that AAV2.5T mediated gene transfer significantly better than its parental serotypes, and corrected the chloride transport defect in CF human airway epithelial cultures. We determined that AAV2.5T developed increased binding to the apical surface of human airway epithelial cells, and that it has evolved to utilize specific 2,3N-linked sialic acid residues on the cell surface that mediate rapid internalization and subsequent infection. Thus, sialic acid serves as not just an attachment factor but is also required for AAV2.5T internalization, possibly representing an important rate-limiting step for other viruses that use sialic acids. Additionally, we utilized directed evolution in vivo in the lungs of pigs to select a novel AAV capsid that is identical to AAV2 except for five point mutations, which we called AAV2H22. We found that AAV2H22 mediated gene transfer to pig airway epithelial cultures significantly better than AAV2, and that it had evolved altered receptor binding. We also found that directed evolution in vitro in human and pig airway epithelial cultures results in the selection of distinct viruses for the two species, and that maintaining different selection stringencies results in the recovery of different AAV variants. Finally, we utilized Hoechst 33342, a DNA binding compound which was previously found to increase AAV transduction in cell lines, to increase AAV-mediated gene expression in primary human airway epithelia. We determined that the mechanism of this effect was due to activation of the CMV promoter. The findings from this research have significant implications for our understanding of AAV biology and for pulmonary gene therapy.

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

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

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.

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

La surdité est le déficit sensoriel le plus fréquent chez l'Homme et touche plus de 360 millions de personnes dans le monde. En France, un enfant sur 700 naît avec une surdité sévère ou profonde, et un enfant sur 1000 deviendra malentendant avant l'âge adulte. Environ 80% des cas de surdité neurosensorielle ont une cause génétique. La surdité peut être associée à des troubles de l'équilibre rendant difficile l'exécution de simples taches quotidiennes. La cause la plus fréquente de déficience auditive et vestibulaire, sont l'atteinte de, respectivement, la cochlée, organe sensoriel de l'audition, et du vestibule, organe sensoriel de l'équilibre, localisés dans l'oreille interne.Face à l'inexistence de traitement curatif, la thérapie génique semble être une alternative afin de traiter les patients atteints de surdités et/ou de troubles vestibulaires héréditaires. L'objectif de mon projet de thèse est de restaurer l'audition et l'équilibre dans des souris modèles des surdités et troubles vestibulaires humains (DFNB9, DFNB59, syndrome de Usher de type 1G et 3A), en utilisant la thérapie génique virale.Les résultats obtenus ont apporté une preuve de principe que le transfert intracochléaire de gènes thérapeutiques contenus dans un adénovirus associé in vivo, permet de restaurer la structure et la fonction des cellules ciliées sensorielles de l'oreille interne, au niveau de l'appareil mécano-sensitif et de la synapse. Ainsi, nous avons restauré de manière significative l'audition, et corriger complètement le trouble vestibulaire. Ce projet ouvre la voie à de nouvelles approches thérapeutiques pour des patients atteints de formes génétiques d'atteinte de l'oreille interne. / Hearing loss is one of the most common human sensory deficits affecting over 360 millions people worldwide. In France, over one child out of 700 suffers from profound deafness at birth, and 1/1000 will be affected by hearing impairment prior to adulthood. The early-onset forms of severe, nonsyndromic deafness are mostly genetic in origin. Deafness can be associated with vestibular impairments which can complicate daily simple tasks. In most cases, hearing and vestibular impairments are due to defects in, respectively, the cochlea, the hearing organ, and the vestibule, the balance organ.In front of the non-existence of curative treatment, gene transfer technology is an alternative therapeutic approach to rescue hereditary deafness and vestibular impairments. The aim of my project is the use of viral gene therapy to restore hearing and balance in mice established as model for human deafness (DFNB9, DFNB59, Usher syndrome type IG and 3A). Our results provide a proof-of-principle that in vivo intracochlear delivery of therapeutic genes using adeno-associated virus can restore the structure and the function of inner ear sensory hair cells, at the mecano-sensitive apparatus and at the synapse. Thus, we restore significantly the hearing, and completely the vestibular impairment. This project open the way to new methods for restoring hearing in patients with genetic forms of deafness.