Дисертації з теми "Adeno-Associated Viral (AAV) vectors"
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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.
Повний текст джерелаTypescript. Title from title page of source document. Document formatted into pages; contains 71 pages. Includes Vita. Includes bibliographical references.
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.
Повний текст джерелаChoudhury, 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.
Повний текст джерелаSteines, 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.
Повний текст джерелаIbraheim, 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.
Повний текст джерелаMoimas, Silvia. "A gene transfer approach, based on Adeno-Associated Viral (AAV) vectors, to study the process of vessel maturation and stabilization." Doctoral thesis, Università degli studi di Trieste, 2009. http://hdl.handle.net/10077/3311.
Повний текст джерелаThe main goal of angiogenic gene therapy is the formation of functional new blood vessels adequate to restore blood flow in ischemic tissues. Angiogenesis is a complex process, consisting in the sprouting of new capillaries from pre-existing vessels to form an immature vascular network, which subsequently undergoes functional maturation and remodelling. Many factors are involved in this process and, among them, the VEGF family members are universally recognized as the key players. During my PhD I exploited gene transfer by vectors based on the Adeno-Associated Virus (AAV) to express several factors involved in the angiogenic process, in an attempt to define the molecular and cellular mechanisms of vessel maturation and stabilization. Most experiments were performed by vector injection in the mouse and rat skeletal muscle, followed by detailed histological, immunohistochemical and functional analysis. First of all the angiogenic effect driven by two main VEGF isoforms, VEGF165 and VEGF121 was compared. AAV-VEGF165 and AAV-VEGF121 appeared equally able to induce endothelial cell proliferation, leading to the formation of new CD31 positive capillaries. However, only the longest VEGF165 isoform was capable to recruit -SMA positive cells around growing capillaries and therefore giving rise to small arteries. The acquisition of a smooth muscle cell layer can be considered as marker of vessel maturation. This was also confirmed by a permeability assay, which showed that VEGF121-induced vessels were more permeable compared to those induced by VEGF165. Interestingly, the presence of -SMA positive vessels was paralleled by the recruitment of CD11b positive mononuclear cells from the bone marrow, cells which were not recruited by VEGF121. The presence of these infiltrating cells in close proximity to the newly formed arterioles suggested their possible role in smooth muscle cell recruitment and vessel maturation. Real-time PCR allowed observing that the infiltrating CD11b positive cells expressed a cocktail of cytokines implicated in vessel maturation, such as TGF- and PDGF-B. As a proof of concept of the paracrine activity of these cells in vessel maturation, we developed an AAV-PDGF-B vector, which, when co-injected with AAV-VEGF121, was arteriogenic even in absence of cellular infiltration. Thus, the expression of PDGF-B partially substitutes for the cells observed in the muscles injected by AAV-VEGF165 to form arterial vessels. To verify the functionality of the vessels induced by AAV-VEGF165 we delivered this vector to different animal models of tissue ischemia: a flap ischemia model and an in vivo chamber for tissue engineering based on an artero-venous loop. In both the models, VEGF165 expression induced the formation of -SMA positive vessels, which turned out to improve flap survival in the flap models, and to promote the formation of new vascularized tissue in the chamber. Despite the presence of several arteries, other vessels formed by VEGF165 were abnormally enlarged and leaky, often forming vascular lacunae. This observation indicated that VEGF gene transfer might not be sufficient for the formation of a fully functional vascular network, and that other factors might be required in order to achieve functional competence of the neovessels. We observed that the combined expression of VEGF165 with Angiopoietin-1, which is known to stabilize endothelial and mural cell interactions, resulted in a significant reduction of vessel permeability and improved blood flow, as assessed by positron emission tomography (PET) and single photon emission tomography (SPECT). These findings reveal that a fine control of the expression of angiogenic factors is needed to achieve the formation of stable and functional vessels. The presence of -SMA positive cells might be considered as a first step in vessel maturation but further stabilization factors have to take part to the process in order to tighten the cell-cell junctions. Moreover, we showed that a detailed histological and functional analysis ex vivo might not be sufficient to characterized the new vasculature, requiring imaging techniques such as PET or SPECT.
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Ruozi, Giulia. "An in vivo functional selection strategy to identify novel genes sustaining cardiac function." Doctoral thesis, Scuola Normale Superiore, 2012. http://hdl.handle.net/11384/85943.
Повний текст джерелаXu, 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.
Повний текст джерелаCarty, Nikisha Christine. "Recombinant AAV Gene Therapy and Delivery." Scholar Commons, 2009. https://scholarcommons.usf.edu/etd/1890.
Повний текст джерелаPacouret, Simon. "Thermostability of Adeno-Associated Virus (AAV) Vectors." Thesis, Nantes, 2018. http://www.theses.fr/2018NANT1041/document.
Повний текст джерелаAdeno-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
Dickey, David Derrick. "Strategies for improving adeno-associated viral infection of airway epithelial cells." Diss., University of Iowa, 2012. https://ir.uiowa.edu/etd/2858.
Повний текст джерелаStachler, 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.
Повний текст джерелаGhosh, Arkasubhra. "Rational design of split gene vectors to expand the packaging capacity of adeno-associated viral vectors." Diss., Columbia, Mo. : University of Missouri-Columbia, 2007. http://hdl.handle.net/10355/4712.
Повний текст джерелаThe entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "December 2007" Includes bibliographical references.
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.
Повний текст джерелаKoo, 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.
Повний текст джерелаPeden, Carmen Elena Socarras. "Characterization of the immune response to recombinant adeno-associated viral vectors in the brain." [Gainesville, Fla.] : University of Florida, 2004. http://purl.fcla.edu/fcla/etd/UFE0004403.
Повний текст джерелаTypescript. Title from title page of source document. Document formatted into pages; contains 134 pages. Includes Vita. Includes bibliographical references.
Ekstedt, Elias, Inna Fryckstedt, Hanna Hyllander, Josefin Jonsson, Elin Ring, and Felix Wærn. "The future of viral vectors for gene therapy." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-444138.
Повний текст джерелаRossi, Axel. "Intracellular fate of AAV particles in human Dendritic Cell and impact on Gene Transfer." Thesis, Lyon, 2016. http://www.theses.fr/2016LYSEN028.
Повний текст джерелаVectors 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
Rohwedder, Carolin [Verfasser], and Martin [Akademischer Betreuer] Müller. "Generation of a shut-off system for Adeno-associated viral gene transfer vectors / Carolin Rohwedder ; Betreuer: Martin Müller." Heidelberg : Universitätsbibliothek Heidelberg, 2017. http://d-nb.info/118098546X/34.
Повний текст джерелаIppodrino, Rudy. "High-throughput RNA interference screening identifies human genes regulating AAV transduction." Doctoral thesis, Scuola Normale Superiore, 2015. http://hdl.handle.net/11384/85970.
Повний текст джерелаOliveira, 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.
Повний текст джерелаO 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.
Schnödt, Maria Angelika [Verfasser], Dagmar [Gutachter] Mörsdorf, Mirka [Gutachter] Uhlirova, and Hildegard [Gutachter] Büning. "Improving safety and establishing episomal maintenance of Adeno-associated viral vectors / Maria Angelika Schnödt ; Gutachter: Dagmar Mörsdorf, Mirka Uhlirova, Hildegard Büning." Köln : Universitäts- und Stadtbibliothek Köln, 2016. http://d-nb.info/1122713827/34.
Повний текст джерелаCataldi, Marcela Patricia. "Diverse Effects of DNA Repair Pathways on the Outcome of Recombinant Adeno-Associated Virus (rAAV) Vector Gene Delivery." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1303842573.
Повний текст джерелаWeinmann, Jonas [Verfasser], and Ana [Akademischer Betreuer] Martin-Villalba. "Massively parallel in vivo characterization of novel adeno-associated viral (AAV) capsids using DNA/RNA barcoding and next generation sequencing / Jonas Weinmann ; Betreuer: Ana Martin-Villalba." Heidelberg : Universitätsbibliothek Heidelberg, 2020. http://d-nb.info/1208222198/34.
Повний текст джерелаSallach, Jessica [Verfasser], Dagmar [Akademischer Betreuer] Knebel-Mörsdorf, and Bent [Akademischer Betreuer] Brachvogel. "Exploiting high-throughput screens to optimize Adeno-Associated Viral Vectors for gene transfer into primary human keratinocytes / Jessica Sallach. Gutachter: Dagmar Knebel-Mörsdorf ; Bent Brachvogel." Köln : Universitäts- und Stadtbibliothek Köln, 2013. http://d-nb.info/1050577019/34.
Повний текст джерелаPagès, i. Pi Gemma. "Intrathecal administration of AAVrh10 coding for β‐glucuronidase corrects biochemical and histological hallmarks of mucopolysaccharidosis type VII mice and improves behavior and survival". Doctoral thesis, Universitat Autònoma de Barcelona, 2015. http://hdl.handle.net/10803/300733.
Повний текст джерелаMucopolysaccharidosis type VII (MPS VII) is an ultrarare monogenic lysosomal storage disease. It is caused by the lack of β-‐glucuronidase activity, a lysosomal enzyme involved in the degradation pathway of glycosaminoglycans. This inborn genetic alteration causes a dysfunction of the lysosomal system that entails abnormal lysosomal storage and disruption of cell homeostasis. The disease presents a range of clinical severity among patients, from death in utero to a life expectancy of up to 20 or 30 years for the milder forms. The severe form of MPS VII among cases with postnatal disease onset is characterized by hepatosplenomegaly, skeletal abnormalities, developmental delay and mental retardation, among other symptoms. Currently, the only treatments for MPS VII patients are interventions to alleviate the symptoms, but no curative treatment is available. Gene therapy is a promising therapeutic approach to find a cure for monogenic diseases. Among the available gene delivery vectors, adeno-‐associated viruses (AAVs) present several features that make them attractive for gene therapy strategies: they are able to transduce dividing and quiescent cells, they provide long term expression of the transgene, they are not able to autonomously replicate without a helper virus, and wild type AAV infections are not pathogenic. Different AAV serotypes have been used as gene therapy vectors in preclinical studies. Among them, AAV9 and AAVrh10 are the serotypes which show greater transduction capacity and a broader range of cell-‐type specific tropism, particularly in the central nervous system. The use of AAVrh10, a non-‐human serotype, may avoid the neutralization by anti-‐AAV immune factors present in human sera after natural AAV infections. However, cross-‐reactivity with antibodies raised against AAV2, the most common human AAV serotype, may still interfere in the therapeutic outcome. In this work, we propose a gene therapy strategy for MPS VII based on a single intrathecal injection of an AAVrh10 coding for the β-‐glucuronidase gene, tested in young adult MPS VII mice. We show that vector delivery to the CSF by lumbar puncture, a poorly invasive technique, allows the transduction of CNS structures using a lower vector dose than by intravenous delivery. In addition, the drainage of the vector from the CSF to the bloodstream results in transduction of somatic organs such as liver, thus providing a systemic β-‐glucuronidase source that achieves serum enzymatic activity comparable to wild type mice. The sustained recombinant enzyme expression by AAV-‐transduced cells, and the cross-‐correction provided by enzyme secretion, attains the correction of biochemical and histopathological hallmarks of the disease in CNS and somatic organs. This correction at the cellular level leads to a significant improvement of physical, cognitive and emotional characteristics of MPS VII mice and a doubling of the MPS VII mouse life span.
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.
Повний текст джерелаGuhasarkar, 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.
Повний текст джерелаAfonso, Inês Torquato. "Biodistribution analysis of striatal and cerebellar administration of modified adeno-associated viral vectors in normal mice." Master's thesis, 2020. http://hdl.handle.net/10400.1/15355.
Повний текст джерелаAdeno-associated viral (AAV) vectors have demonstrated to be safe and reliable gene delivery systems to the central nervous system (CNS). Due its immune privileged status, it is difficult to deliver the therapeutical treatment to the target cell and treat neurological disorders of the CNS. This study investigates the spreading, transduction, cell tropism, and toxicity of a novel mosaic AAV, the AAV1/AAV2 delivery system, upon intraparenchymal injection in the striatum and in the deep cerebellar nuclei (DCN) of normal mice. The mosaic AAV1/AAV2 vector was produced using the “triple” transfection method, however, the packing cell was transduced by 2 plasmids encoding for the AAV1 and AAV2 serotype capsid proteins instead of one. The first cohort of C57BL/6 mice was bilaterally injected with 3×109 vg in the striatum with mosaic AAV1/AAV2 or parental serotypes, AAV1 and AAV2. The second cohort was bilaterally injected in the DCN region of the cerebellum with 5×109 vg of mosaic AAV1/AAV2 or parental serotype. The results indicate that in the striatal injection, the mosaic AAV1/AAV2 vector had better spreading and transduce more brain regions than the parental serotypes. The mosaic AAV1/AAV2 and parental vector tropism was unable to be detected with the used antibodies, NeuN, GFAP and Iba-1, which detect neurons, astrocytes, and microglia, respectively, in both striatum and DCN. None of the viral vectors exhibited toxicity in the striatal injection. In the DCN injection, the AAV1 serotype had better spreading and higher transduction levels than the other vectors. In conclusion, the mosaic AAV1/AAV2 is a safe delivery vehicle for CNS with wide transduction results upon intraparenchymal injection. It is capable of sustaining the AAV1 neuronal tropism, as well as to be purified by the AAV2 standard technique, i.e. heparin affinity chromatography. However, further investigation is necessary to increase the sample size and determine the vector cell tropism.
Cheng, Yu-Lei. "Investigation of Sf-9 Cell Metabolism Before and After Baculovirus Infection Using Biovolume: a Case for the Improvement of Adeno-Associated Viral Vector Production." Thesis, 2009. http://hdl.handle.net/10012/4540.
Повний текст джерелаPerabò, Luca [Verfasser]. "Adeno-associated virus display : in vitro evolution of AAV retargeted vectors / Luca Perabò." 2003. http://d-nb.info/972496246/34.
Повний текст джерелаQureshi, Wajeeha Rashid. "Adeno-associated viral vectors for tissue-specific gene delivery in vivo." Thesis, 2017. https://hdl.handle.net/2144/23998.
Повний текст джерелаShevtsova, Zinayida. "Development of Viral Tools for CNS Gene Transfer: Adeno-Associated Viral Vectors in Gene Therapy of Parkinson's Disease." Doctoral thesis, 2006. http://hdl.handle.net/11858/00-1735-0000-0006-B5F2-9.
Повний текст джерелаCarreiras, Rafael Baganha. "Development of Single-Step Affinity Chromatography Protocols for the Purification of Adeno-Associated Viral Vectors." Master's thesis, 2020. http://hdl.handle.net/10316/92998.
Повний текст джерелаNas últimas décadas, os vetores Virais Adeno-Associados (VAAs) têm-se destacado como um sistema de entrega promissor na área da terapia génica. De facto, vários estudos pré-clínicos demonstraram um conjunto de propriedades únicas deste tipo de vírus, incluindo um perfil robusto de segurança, uma transdução eficiente e uma baixa imunogenicidade. Foram ainda reportadas outras características relevantes, como a expressão prolongada dos seus transgenes em diversos órgãos, assim como um tropismo seletivo dos diferentes serótipos. Tendo tudo isto em consideração, a comunidade científica sentiu-se confiante em explorar o uso dos VAAs recombinantes num contexto clínico; daí que, actualmente, mais de 100 estudos clínicos usando VAAs estão em curso. No entanto, apesar dos resultados promissores obtidos em vários ensaios clínicos, há uma necessidade emergente para o desenvolvimento e otimização de protocolos de produção e purificação que possibilitem o fabrico de vetores VAAs altamente puros e em grande escala. Assim, o principal objetivo deste estudo foi simplificar e melhorar a purificação de vetores VAAs, através do desenvolvimento de protocolos simples e eficientes, baseados em cromatografia, que tirem partido das propriedades bioquímicas naturais dos diferentes serótipos de VAAs. Deste modo, vetores virais do tipo mosaico (rVAA1/2) foram purificados utilizando três colunas de cromatografia de afinidade distintas e os respetivos protocolos experimentais otimizados. A eficiência das três estratégias de purificação foi avaliada através da caracterização dos rVAAs purificados, em termos de pureza, propriedades físicas e atividade biológica. Os resultados obtidos demonstraram que os três protocolos de purificação foram eficientes na obtenção de vetores virais com elevado título e grau de pureza, num sistema passível de ser usado em larga escala. Adicionalmente, os stocks purificados de vetores rVAA1/2 evidenciaram resultados promissores em experiências de transfeção in vitro, relevando-se capazes de transfetar com sucesso linhas primárias, bem como de expressar o gene de interesse. Em resumo, este estudo fornece evidências claras da rapidez, eficiência e potencialidade para o seu uso em grande escala dos métodos baseados em colunas de cromatografia de afinidade para a purificação de rVAAs, diretamente dos lisados de células produtoras, com uma relação custo-benefício atrativa.
Over the past decades, Adeno-Associated Viruses (AAVs) have arisen as a promising delivery system for human gene therapy. Indeed, several in vitro and in vivo preclinical studies have already shown a handful of properties which are unique to this kind of viruses, including a strong safety profile, high gene transfer efficiency and low immunogenicity. Moreover, a prolonged gene expression in several tissues as well as a selective tissue tropism has also been reported. Having all the above in mind, the scientific community felt compelled to resource to the use of recombinant AAVs (rAAVs) for therapeutic gene transfer into patients in the clinical setting, with more than one hundred studies using AAVs currently taking place. However, despite the promising results obtained from several clinical trials, it has also become clear the emerging need for the development of production and purification protocols for the manufacturing of large amounts of highly pure rAAV vectors.Therefore, the main goal of this project was to simplify and improve rAAV vector purification by the establishment of simple, but efficient chromatography-based protocols, taking advantage of the natural biochemical properties of AAV serotypes.In order to do this, mosaic rAAV1/2 vectors were purified using three affinity chromatography columns and the efficiency of the different strategies was evaluated through the characterization of the purified rAAVs in terms of purity, physical properties and biological activity.Overall, the obtained results show that our scalable purification protocols were efficient in obtaining AAV vectors with high titer and purity. Moreover, the purified rAAV1/2 stocks have been successfully used in in vitro transfection experiments.In summary, this study provides compelling evidence of fast, efficient, cost effective, and scalable column-based methods for large-scale rAAV purification of various recombinant adeno-associated viruses directly from the lysates of producer cells.
Outro - This work was funded by the ERDF through the Regional Operational Program Center 2020, Competitiveness Factors Operational Program (COMPETE 2020, POCI) and National Funds through FCT (Foundation for Science and Technology) - BrainHealth2020 projects (CENTRO-01-0145-FEDER-000008), UID/NEU/04539/2019, ViraVector (CENTRO-01-0145-FEDER-022095), CortaCAGs (PTDC/NEU-NMC/0084/2014|POCI-01-0145-FEDER-016719), SpreadSilencing POCI-01-0145-FEDER-029716, Imagene POCI-01-0145-FEDER-016807, CancelStem POCI-01-0145-FEDER-016390, POCI-01-0145-FEDER-030737, POCI-01-0145-FEDER-032309, as well as SynSpread, ESMI and ModelPolyQ under the EU Joint Program - Neurodegenerative Disease Research (JPND), the last two co-funded by the European Union H2020 program, GA No.643417; by National Ataxia Foundation (USA), the American Portuguese Biomedical Research Fund (APBRF) and the Richard Chin and Lily Lock Machado-Joseph Disease Research Fund.
Maina, Caroline Njeri. "Obstacles and Circumvention Strategies for Hematopoietic Stem Cell Transduction by Recombinant Adeno-associated Virus Vectors." Thesis, 2009. http://hdl.handle.net/1805/1869.
Повний текст джерелаHigh-efficiency transduction of hematopoietic stem cells (HSCs) by recombinant adeno-associated virus serotype 2 (AAV2) vectors is limited by (i) inadequate expression of cellular receptor/co-receptors for AAV2; (ii) impaired intracellular trafficking and uncoating in the nucleus; (iii) failure of the genome to undergo second-strand DNA synthesis; and (iv) use of sub-optimal promoters. Systematic studies were undertaken to develop alternative strategies to achieve high-efficiency transduction of primary murine HSCs and lineage-restricted transgene expression in a bone marrow transplant model in vivo. These included the use of: (i) additional AAV serotype (AAV1, AAV7, AAV8, AAV10) vectors; (ii) self-complementary AAV (scAAV) vectors; and (iii) erythroid cell-specific promoters. scAAV1 and scAAV7 vectors containing an enhanced green-fluorescent protein (EGFP) reporter gene under the control of hematopoietic cell-specific enhancers/promoters allowed sustained transgene expression in an erythroid lineage-restricted manner in both primary and secondary transplant recipient mice. Self complementary AAV vectors containing an anti-sickling human beta-globin gene under the control of either the beta-globin gene promoter/enhancer, or the human parvovirus B19 promoter at map-unit 6 (B19p6) were tested for their efficacy in a human erythroid cell line (K562), and in primary murine hematopoietic progenitor cells (c-kit+, lin-). These studies revealed that (i) scAAV2-beta-globin vectors containing only the HS2 enhancer are more efficient than ssAAV2-beta-globin vectors containing the HS2+HS3+HS4 enhancers; (ii) scAAV-beta-globin vectors containing only the B19p6 promoter are more efficient than their counterparts containing the HS2 enhancer/beta-globin promoter; and (iii) scAAV2-B19p6-beta-globin vectors in K562 cells, and scAAV1-B19p6-beta-globin vectors in murine c-kit+, lin- cells, yield efficient expression of the beta-globin protein. These studies suggest that the combined use of scAAV serotype vectors and the B19p6 promoter may lead to expression of therapeutic levels of beta-globin gene in human erythroid cells, which has implications in the potential gene therapy of beta-thalassemia and sickle cell disease.
Burt, Daniel Robert. "Optimization of viral transduction in the central nervous system." Thesis, 2014. https://hdl.handle.net/2144/14650.
Повний текст джерелаShevtsova, Zinayida [Verfasser]. "Development of viral tools for CNS gene transfer : adeno-associated viral vectors in gene therapy of Parkinson's disease / submitted by Zinayida Shevtsova." 2006. http://d-nb.info/982407254/34.
Повний текст джерелаBourhill, Tarryn. "Successful disabling of the 5' UTR of HCV using adeno-associated viral vectors to deliver artificial primary microrna mimics." Thesis, 2015. http://hdl.handle.net/10539/18687.
Повний текст джерелаChronic hepatitis C virus (HCV) infection is a major health concern and is strongly associated with cirrhosis, hepatocellular carcinoma and liver related mortality. The current standard treatment for HCV is a combination of interferon-based therapies and ribavirin, which only produces sustained viral suppression in 40-50% of patients. Thus, the development of new treatments for HCV infection is critical. The HCV genome is the template for both protein translation and viral replication and, being RNA, is amenable to direct genetic silencing by RNA interference (RNAi). HCV is a highly mutable virus with error prone RNA replication and it has been previously reported that the virus can escape RNAi-mediated treatments through various point mutations. This has highlighted the importance of developing RNAi-based therapy that simultaneously targets multiple regions of the HCV genome. Thus, five artificial primary miRNA (pri-miRNA) were designed to mimic the naturally occurring monomeric pri-miRNA-31. The natural guide sequence on the 5’ arm of the pri-miRNA-31 was replaced with sequences complementary to different regions of the 5’ UTR of HCV. Potent knockdown of an HCV reporter was seen with four of the five constructs, and these were used to generate polycistronic cassettes, which showed impressive silencing of an HCV target. To further their application as a gene therapy recombinant adeno-associated viral (rAAV) vectors that express the polycistronic pri-miRNA mimics were generated. Two different promoter sequences were used to direct the expression of the polycistronic constructs. Ubiquitously expressed CMV and liver-specific mTTR promoters were used to generate rAAVs. All of the vectors enter liver- derived cells efficiently and significantly knock down the expression of an HCV target and showed dramatic inhibition of HCV replicon replication. The expressed polycistronic pri-miRNA mimics did not induce any off-target effects, such as stimulation of the immune response and saturation of the RNAi pathway. All the pri-miRNA mimics within the polycistronic cassettes were processed according to their intended design. The anti-HCV rAAVs developed have the potential to be an effective therapy that may contribute to the eradication of HCV.
Huang, Yung An, and 黃永安. "Danger Signal Extracellular ATP Is a Target For Adeno-associated Viral Vectors Mediated Gene Therapy in a Murine Asthma Model." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/38077135092117679109.
Повний текст джерела長庚大學
生物醫學研究所
101
Asthma is a chronic respiratory disease characterized by recurrently attacks of breathlessness and wheezing. Th2 response plays a crucial role in pathogenesis of asthma, and results in eosinophilia and airway hyper-responsiveness. ATP accumulation in bronchoalveolar lavage fluid (BALF) of asthmatic subjects indicates that extracellular ATP and the downstream responses are involved. ATP is able to escape from cells through functional pannexin-1, a nonselective pore formation channel associated with P2X7 receptor. Furthermore, ATP is hydrolyzed to AMP by ubiquitous ecto-ATP/ADPase, CD39, and the expression of CD39 is downregulated during inflammation. Our study indicated that extracellular ATP levels were upregulated in inflammatory mediator-stimulated human epithelial cell lines and mouse macrophage cell lines. The quantity of extracellular ATP was reduced by pannexin-1 inhibitor carbenoxolone. We subsequently applied an OVA-induced asthma murine model for further experiments. An elevated ATP concentration in BALF of sensitized mice was emerged, and RNA expression levels of CD39 were decreased in the lungs of sensitized mice compared with normal controls. For applying gene therapy to target the signaling of extracellular ATP, pannexin-1 inhibitory mimetic peptide 10panx1 was administered to OVA-sensitized mice, leading to reduced IL-5 production. These data suggested that panaexin-1 and CD39 were potential targets for gene therapy. We applied secretory form of 10panx1 and CD39 to adeno-associated viral (AAV) vector expression system to deliver the coding sequences to the lungs. In experiments of AAV-10panx1, the data showed a decreased level of BALF ATP, asthmatic eosinophilia, IL-5 production from lymph node cells, and OVA-specific IgE. In comparison with vector controls, it revealed only a modulated asthmatic eosinophilia. In the experiments of AAV-CD39, RNA expression level of CD39 was rescued. BALF ATP, asthmatic eosinophilia, IL-5 production from lymphocytes, and OVA-specific IgE were decreased comparing to sensitized controls. When in comparison with vector controls, it only showed a downregulation of asthmatic eosinophilia and OVA-specific IgE. These two treatments partially attenuated the asthmatic parameters. Thus, we considered that these two genes could be the potential targets for gene therapy regarding to allergic disease or asthma.
Lopes, Miguel Monteiro. "AAV-Based Tools For The Development Of Cellular and Animal Models of Machado-Joseph Disease." Master's thesis, 2017. http://hdl.handle.net/10316/81395.
Повний текст джерелаA doença de Machado-Joseph (DMJ), também conhecida como ataxia espinocerebelosa do tipo 3 é uma doença neurodegenerativa caracterizada por uma expansão anormal do tripleto CAG na região codificante do gene MJD1/ATXN3, o que se traduz numa expansão de uma cadeia de poliglutaminas na proteína ataxina-3. Esta expansão de CAGs anormalmente longa confere toxicidade à proteína ataxina-3 produzida que através de múltiplos mecanismos patogénicos culminando em neurodegenerescência em diversas regiões do cérebro.As abordagens terapêuticas atuais consistem principalmente em sessões de fisioterapia e no alívio farmacológico sintomático de sintomas específicos, constituindo a força motriz para o desenvolvimento de novas abordagens terapêuticas.Os diversos modelos existentes de DMJ têm sido ferramentas indispensáveis para a identificação dos mecanismos intrínsecos da doença, bem como para a validação de novas terapias. No entanto, modelos transgénicos de murganho são altamente dispendiosos, necessitam de longos períodos para o desenvolvimento de fenótipo, não recapitulando algumas das características desta doença. O nosso grupo foi pioneiro no desenvolvimento de um modelo roedor da DMJ com base em vetores lentivirais. Apesar das inúmeras vantagens deste modelo, este envolve intervenção cirúrgica (craniotomia), injeção localizada no parênquima cerebral, estando a patologia confinada ao local de injeção. Estas evidências demonstram a urgência no desenvolvimento de novos modelos que expressem ataxina-3 mutante de forma ubíqua no Sistema Nervoso Central (SNC), providenciando novas perspetivas relacionadas com os mecanismos da doença, permitindo ainda a avaliação do potencial de novas terapias.O rápido desenvolvimento de ferramentas baseadas em Vírus Adeno-Associados (AAV), tornou-se um dos mais promissores sistemas de entrega de genes a uma grande variedade de tipos celulares, através de diferentes vias de administração.O objetivo do presente trabalho foi o desenvolvimento de novas estratégias baseadas no uso de vetores mosaico de AAV para gerar modelos in vitro e in vivo de DMJ. Para tal, foram desenvolvidos vetores mosaico AAV1/2 e AAV2/9 codificando para a ataxina-3 humana mutada direcionados para o SNC. Esta abordagem providenciou o desenvolvimento de modelos de DMJ com elevada relevância fisiológica em tempo-útil e com uma boa relação custo-benefício, ultrapassando algumas das limitações mencionadas anteriormente.Resumidamente, este estudo fornece forte evidências que os vetores AAVs gerados são capazes de transduzir o SNC após injeção intracraniana (AAV1/2) ou intravenosa (AAV2/9), sobre expressando a ataxina-3 mutante completa não só em modelos in vitro como também in vivo, recapitulando algumas das principais características da DMJ.
Machado-Joseph Disease (MJD) or Spinocerebellar Ataxia type 3 (SCA3) is a neurodegenerative disorder characterized by an abnormal expansion of the CAG triplet in the coding region of MJD-1/ATXN3 gene, translating into an expanded polyglutamine tract within the ataxin-3 protein. This abnormally long CAG expansion, confers a toxic gain of function to the ataxin-3 protein that through multiple pathogenic mechanisms leads to neurodegeneration in several brain regions. Current therapeutic approaches consist mainly in the use of physiotherapy and in the pharmacological alleviation of specific symptoms, thus encouraging further investigation towards possible therapeutic approaches.The several existing models of MJD have been useful tools that largely contributed to the identification of intrinsic pathways affecting the disease as well as the validation of new therapies. However, transgenic mouse models are expensive, take long periods of time to develop a phenotype, or do not recapitulate some of the hallmarks of this disease. Our group was pioneer in developing cost-effective lentiviral-based rodent models of MJD. Despite the numerous advantages of this model, it involves craniotomy, in situ injection in the brain parenchyma and the pathology is only confined to the local of injection. In light of these evidences, there is an urgent need for new mouse models that widely express mutant ataxin-3 throughout the Central Nervous System (CNS), potentially providing new insights into the disease mechanisms and allowing screening of novel therapies.The rapidly expanding Adeno-Associated Virus (AAV) vector toolkit has become one of the most promising viral vectors delivering genetic cargo to a wide range of cell types through different routes of administration. In the present work, we aimed to develop new strategies based on the use of mosaic rAAV vectors, to generate in vitro and in vivo models of MJD. For that purpose, mosaic vectors AAV1/2, and AAV2/9 encoding the mutant human ataxin-3 have been developed to efficiently target and transduce the CNS. This approach provided physiologically relevant, time-effective, and cost-effective models for MJD, circumventing some of the limitations of above-mentioned models.In summary, this study provides compelling evidence that the generated mosaic rAAVs are able to efficiently transduce the CNS upon intracranial (AAV1/2) or intravenous injection (AAV2/9), and overexpress full-length mutant ataxin-3 both in vitro and in vivo, recapitulating some of the hallmarks of MJD.
FCT
Outro - American Portuguese Biomedical Research Fund (APBRF)
Outro - BrainHealth2020 (CENTRO-01-0145-FEDER-000008)
Outro - CortaCAGs (POCI-01-0145-FEDER-016719)
Outro - H2020 da União Europeia, GA No. 643417
Outro - “National Ataxia Foundation” (USA)
Outro - POCI-01-0145-FEDER-007440
Outro - Richard Chin and Lily Lock Machado Joseph Disease Research Fund
Outro - ViraVector (CENTRO-01-0145-FEDER- 022095)
Wang, Chi-Hsien, and 王啟賢. "The application of Adeno-associated viral vectors and the interference RNA technique to generated the mouse animal model of Limb-Girdle Muscular Dystrophy 2I." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/27986403192731142029.
Повний текст джерела國防醫學院
醫學科學研究所
99
Limb-girdle muscular dystrophy 2I (LGMD2I) is caused by genetic mutations in the gene encoding fukutin related protein (FKRP). Unlike its severe allelic forms such as congenital muscular dystrophy 1C (CMD1C), Walker-Warburg Syndrome (WWS) and muscle-eye-brain disease (MEB), LGMD2I usually shows slower onset and develops milder course without central nervous system defects. Currently there is a lack of viable animal models that closely recapitulate LGMD2I clinical phenotypes. In this thesis, I used RNAi technology to knockdown FKRP expression via postnatal gene delivery to circumvent embryonic lethality. Here, I try to utilize the adeno-associated virus (AAV) which is considered an efficient, convincing and robust gene delivery vector transfered the short hairpin (shRNA) genes to healthy ICR mice. AAV vectors carrying either a single cassette expressing one of the two pre-screened shRNAs, or a dual cassette expressing both shRNAs, were delivered into the hind limb muscles by a one-time intramuscular injection. We showed that FKRP expression at 10 months post-injection was reduced by 50% with both shRNA FKRP2 and shRNA FKRP5, and 75% with the dual cassette shRNA FKRP2+5. Glycosylation of -dystroglycan was also significantly reduced. Dystrophic pathology including central nucleation in >50% of the myofibers and the elevation of creatine kinase activity became evident at 10 months, but not at 2 months, post-injection of the dual shRNA FKRP2+5 cassette. These results suggest that a reduction of approximately 75% of the normal level of FKRP expression is required to induce chronic dystrophic phenotypes in skeletal muscles. The results further imply that greater than 25% of the normal FKRP level is necessary for LGMD2I therapy to effectively correct the genetic deficiency and prevent dystrophic pathology. To evaluate both of the systemic AAV-delivered FKRP knock-down mice and FKRP L276I missense knock-in mice, I also found the same pathological features which were found on the LGMD2I patients, i.e. central nucleation, the insufficient glycosylation of the -dystroglycan and the cardiac conductive abnormality-left bundle branch block (LBBB). But it’s valuable to mention that the homozygous FKRP L276I mutation mice revealed more sever symptoms than the AAV-mediated mice. This finding suggested that the FKRP expression level might be a crucial point for the treatment studies of the LGMD2I.