La littérature décrit des différences fondamentales entre l’AAV2 sauvage et ses pendants recombinants. La forme sauvage serait plus efficiente en terme de production, d’encapsidation et d’infectieusité, allant de facteurs de deux à cents en fonction de la propriété étudiée. A cause de ces différences, la quantité de rAAV nécessaire pour traiter un patient atteint d’une maladie implique une injection systémique estimée à 1.1015 particules par kilogramme de tissue à traiter. C’est dans cette optique que s’inscrivent mes travaux de thèse. Essayer de comprendre qu’elles sont les différences entre l’AAV sauvageet les rAAV qui peuvent engendrer tant de différences en terme d’efficacité. L’étude du comportement du génome de l’AAV2 sauvage dans le système baculovirus/cellules Sf9, a permis de découvrir que la régulation de l’AAV2 sauvage était similaire en baculovirus. Nous avons aussi découvert, grâce à notre analyse transcriptomique que les promoteurs naturels del’AAV2 étaient actifs dans notre système, ce qui nous a permis d’imaginer de nouvelles constructions génétiques afin d’améliorer la quantité et la qualité des particules rAAV. Nous avons aussi réaliser des études structurales sur différentesparticules AAV afin d’améliorer notre connaissance de ces particules. / The literature describes several fundamental differences between WT AAV and rAAP properties. WT form obtains an one hundred higher production yield compared to rAAV, with the possibility to obtain only full particles, and most importantly, all the WT AAV particles are infectious, compared to only 1% for the rAAV. These lower values for rAAV, implied to inject up to 1.1015 particles per kilogram of tissue. These quantities induce a non-negligible cost for rAAV based gene therapies, even with the productions techniques improvements or the development of baculoviruses based techniques. It is with these ideas in mind that my PhD works were developed. Trying to understand differences between WT and rAAV, trying to produce WT AAV in baculovirus has bring important knowledges about AAV comportment in baculovirus. Our RNA-Seq results have demonstrated than the WT AAV natural promoters were all active, allowing us to design to genetic constructs in order to improve rAAV quantity and quality. We have also tried to solve the AAV crystal structure, to improve our knowledges about these particles.

Adeno-associated virus type 2 (AAV-2) integrates preferentially into a defined site on human chromosome 19, and has been developed as a gene therapy vector. We propose to use this unique recombination event for site-specific integration of transgenes in plants. This strategy would alleviate problems associated with current plant transformation methods that integrate transgenes randomly. The AAV-2 gene encoding the enzyme that catalyzes the insertion (rep78) was introduced into Arabidopsis thaliana via Agrobacterium-mediated transformation. PCR and sequence analysis confirmed the presence of rep78 in two plant lines. RT-PCR demonstrated rep78 transcription in one plant line, but protein could not be detected in either line. / Master of Science

No description available.

Multiple DNA transactions are at the center of almost all processes regulating the AAV life cycle. A common feature shared by all transactions is the binding of the large AAV Rep proteins Rep78/Rep68 onto DNA sites harboring multiple GCTC repeats. AAV mediated site-specific integration is contingent upon the formation of a productive complex between Rep78/Rep68 and the AAVS1 site located at chromosome 19. In order to understand the mechanistic details of the initial assembly process we carried out equilibrium binding experiments of Rep68 and its individual domains with a 42-mer AAVS1 site. Results show that although Rep68 binds AAVS1 with high affinity (69 nM), both the OBD and helicase individual domains bind DNA weakly with affinities of >>60μM and 22μM respectively under our experimental conditions. Mutant Rep68 proteins that have a defective oligomerization interface bind DNA poorly suggesting that productive binding requires both the concerted interaction of the individual domains with DNA and oligomerization. Moreover, we show that a minimal number of two repeats is required to form a stable complex. In addition, initial studies were done to characterize the interaction between Rep68 and the viral ITR DNA sequences using AUC and electron microscopy.

The transfer of a desired gene to several types of target tissues has been accomplished successfully in the past using existing Adeno-associated viruses (AAVs). Also, it has recently been shown that AAV can stimulate robust antibody responses due to long-term transgene expression or abolishment of transgene product by cell-mediated immune responses, suggesting the potential use of AAVs as vaccines. Most humans already have pre-existing immunity to common AAV serotypes making novel AAVs of low seroprevalence attractive as gene transfer or vaccine vehicles. This thesis describes my primary research objectives that included the isolation of novel AAV serotypes based on AAV DNA sequences from porcine tissues, novel AAV vector production, and biological characterization of porcine AAVs in vitro and in vivo. This was followed by evaluating immune responses in mice vaccinated with porcine AAV vectors expressing the hemagglutinin (HA) from the avian influenza A/Hanoi/30408/2005 (H5N1) strain. These findings show that low seroprevalence porcine AAV vectors were able to efficiently transduce a wide range of cells and tissues. The porcine vectors also performed well as vaccine candidates and were efficient at stimulating host immune responses. Although porcine vectors were successful as vaccines, further studies involving long term gene expression by porcine AAVs is still necessary to confirm their role as gene therapy vehicles.

The transfer of a desired gene to several types of target tissues has been accomplished successfully in the past using existing Adeno-associated viruses (AAVs). Also, it has recently been shown that AAV can stimulate robust antibody responses due to long-term transgene expression or abolishment of transgene product by cell-mediated immune responses, suggesting the potential use of AAVs as vaccines. Most humans already have pre-existing immunity to common AAV serotypes making novel AAVs of low seroprevalence attractive as gene transfer or vaccine vehicles. This thesis describes my primary research objectives that included the isolation of novel AAV serotypes based on AAV DNA sequences from porcine tissues, novel AAV vector production, and biological characterization of porcine AAVs in vitro and in vivo. This was followed by evaluating immune responses in mice vaccinated with porcine AAV vectors expressing the hemagglutinin (HA) from the avian influenza A/Hanoi/30408/2005 (H5N1) strain. These findings show that low seroprevalence porcine AAV vectors were able to efficiently transduce a wide range of cells and tissues. The porcine vectors also performed well as vaccine candidates and were efficient at stimulating host immune responses. Although porcine vectors were successful as vaccines, further studies involving long term gene expression by porcine AAVs is still necessary to confirm their role as gene therapy vehicles.

No description available.

Orientador: Francisco Benedito Teixeira Pessine / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-18T17:10:14Z (GMT). No. of bitstreams: 1 Baldim_Victor_M.pdf: 1808226 bytes, checksum: 7d81445a4d4297b7466e764b52804247 (MD5) Previous issue date: 2011 / Resumo: Neste trabalho preparamos e utilizamos nanopartículas de PLGA-DOTAP encapsulando o peptídeo P17, que bloqueia a ação da citocina imunossupressora TGF-b, no tratamento de camundongos BALB/c fêmea, inoculados via subcutânea com células de hepatocarcinoma (CHC) murino BNL ou de câncer colo-retal (CCR) murino CT26. No modelo de CHC, nanopartículas foram administradas em conjunto com vírus adenoassociados recombinantes, cujo DNA viral codificava a citocina imunoestimuladora IL-12. No modelo de CCR, nanopartículas foram testadas como potencializadoras de uma vacina contra câncer colo-retal. Além disso, a biodistribuição de nanopartículas de PLGA-DOTAP, funcionalizadas ou não com a glicoproteína assialofetuína, foi verificada pelos métodos de bioluminescência em câmara escura com detector CCD e por PCR quantitativa em tempo real. Como resultado, observou-se que nanopartículas de PLGA-DOTAP encapsulando o fármaco P17 são capazes de potencializar a vacina contra CCR e que a glicoproteína assialofetuína é capaz de direcioná-las ao fígado / Abstract: In this research we prepared and used PLGA-DOTAP nanoparticles loaded with the peptide P17, which blocks the action of the imunossupressive cytokine TGF-b, in the treatment of female BALB/c mice inoculated subcutaneously with BNL (murine hepatocellular carcinoma (HCC) cells) or CT26 (murine colorectal cancer (CRC) cells). In the HCC model, nanoparticles were administered together with recombinant adeno-associated virus whose viral DNA encodes the immunostimulatory cytokine IL-12. In the CRC model, nanoparticles were tested as vaccine booster against colorectal cancer. In addition, the biodistribution of PLGA-DOTAP nanoparticles functionalized or not with the asialofetuin glycoprotein was verified by bioluminescence and real-time quantitative PCR. As a result, it was observed that P17 containing DOTAP-PLGA nanoparticles are able to increase vaccine efficiency and that asialofetuin glycoprotein is able to direct them to the liver / Mestrado / Físico-Química / Mestre em Química

Adeno-associated virus (AAV) is a 25 nm replication deficient DNA virus most commonly studied for human gene therapy applications. The work detailed in this thesis investigates the controlled delivery of AAV from surfaces for both transduction and biosensing applications. First, AAV was tested for compatibility with substrate-mediated gene delivery approaches. Two reverse transduction applications were investigated: (1) spatial localization of cells and virus vectors for tissue engineering applications and (2) live cell genetic microarrays. To drive the proper differentiation and assembly of cells within tissue engineering constructs, gene expression patterns may need to be tightly regulated. To localize adhesive proteins and AAV, polydimethylsiloxane stamps and protein adhesive alkanethiols were used. By adsorbing AAV onto adhesive proteins, including human fibronectin, laminin, collagen I, elastin and poly-l-lysine, both cell adhesion and gene delivery were localized to a defined pattern. Gene delivery was efficient on all protein surfaces, with higher expression observed on laminin surfaces. AAV was also patterned using a robotic spotter to create live cell genetic microarrays, creating localized cell islands expressing GFP. This potentially high-throughput technique could be extended to study complex genetic interactions within cells, such as stem cells or induced pluripotent stem cells. Additionally, AAV was explored as a biosensor by modifying virus output functionalities. Wild-type AAV2 externalizes an N-terminus region containing a phospholipase A2 (PLA2) domain during intracellular processing, allowing the virus to escape the endosomal pathway and deliver genetic cargo. This externalization can be replicated outside of cells through heat treatment. AAV2-ΔPLA2-His was created by replacing the PLA2 domain with a nickel binding hexahistidine tag. This replacement allows heat-treated mutant virus to bind a nickel affinity column. Finally, directed evolution was used to (1) improve the ability of AAV to deliver genes into target cells or (2) alter AAV biosensor inputs. Virus libraries were created using error-prone polymerase chain reaction (EP-PCR) to introduce random amino acid modifications into the protein capsid. The error rate for these libraries was estimated to be between 5-7 errors per cap gene. Combining surface immobilization with directed evolution could allow for precise control of AAV for gene delivery and biosensing.

Spinal muscular atrophy is caused by mutation of the SMN1 gene resulting in the selective loss of spinal cord motor neurons. HuD has been shown to interact with SMN and to localize to RNA granules along axons. In conditions where SMN is decreased, like in SMA, HuD’s localization to RNA granules affected. Overexpression of HuD in an SMA cell culture model was shown to rescue SMA-like axonal defects. Here, existence of a signaling pathway downstream of PKC leading to the activation of HuD was investigated in MN-1 cells. Stimulation of this pathway using a pharmacological agonist of PKC increased HuD levels and enhanced its binding to GAP-43 and Tau mRNAs. An scAAV9 viral expression system to overexpress HuD in vivo was established, laying the foundation for the next phase of the study. Overall, modulating HuD expression and activity would be beneficial and could constitute an attractive therapeutic approach for SMA.