Thèses : « Adeno-Associated Viral (AAV) vectors »

1

Lauramore, Amanda K. « Retinal cell tropism of adeno-associated viral (aav) vector serotypes ». [Gainesville, Fla.] : University of Florida, 2004. http://purl.fcla.edu/fcla/etd/UFE0005301.

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Thesis (M.S.)--University of Florida, 2004.
Typescript. Title from title page of source document. Document formatted into pages; contains 71 pages. Includes Vita. Includes bibliographical references.

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Choudhury, Sourav Roy. « Developing an Adeno-Associated Viral Vector (AAV) Toolbox for CNS Gene Therapy : A Dissertation ». eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/809.

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Neurological disorders – disorders of the brain, spine and associated nerves – are a leading contributor to global disease burden with a sizable economic cost. Adeno-associated viral (AAV) vectors have emerged as an effective platform for CNS gene therapy and have shown early promise in clinical trials. These trials involve direct infusion into brain parenchyma, an approach that may be suboptimal for treatment of neurodegenerative disorders, which often involve more than a single structure in the CNS. However, overall neuronal transduction efficiency of vectors derived from naturally occurring AAV capsids after systemic administration is relatively low. We have developed novel capsids AAV-AS and AAV-B1 that lead to widespread gene delivery throughout the brain and spinal cord, particularly to neuronal populations. Both transduce the adult mouse brain >10-fold more efficiently than the clinical gold standard AAV9 upon intravascular infusion, with gene transfer to multiple neuronal sub-populations. These vectors are also capable of neuronal transduction in a normal cat. We have demonstrated the efficacy of AAV-AS in the context of Huntington's disease by knocking down huntingtin mRNA 33-50% after a single intravenous injection, which is better than what can be achieved by AAV9 at the particular dose. AAVB1 additionally transduces muscle, beta cells, pulmonary alveoli and retinal vasculature at high efficiency, and has reduced sensitivity to neutralizing antibodies in human sera. Generation of this vector toolbox represents a major step towards gaining genetic access to the entire CNS, and provides a platform to develop new gene therapies for neurodegenerative disorders.

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Choudhury, Sourav Roy. « Developing an Adeno-Associated Viral Vector (AAV) Toolbox for CNS Gene Therapy : A Dissertation ». eScholarship@UMMS, 2001. http://escholarship.umassmed.edu/gsbs_diss/809.

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Neurological disorders – disorders of the brain, spine and associated nerves – are a leading contributor to global disease burden with a sizable economic cost. Adeno-associated viral (AAV) vectors have emerged as an effective platform for CNS gene therapy and have shown early promise in clinical trials. These trials involve direct infusion into brain parenchyma, an approach that may be suboptimal for treatment of neurodegenerative disorders, which often involve more than a single structure in the CNS. However, overall neuronal transduction efficiency of vectors derived from naturally occurring AAV capsids after systemic administration is relatively low. We have developed novel capsids AAV-AS and AAV-B1 that lead to widespread gene delivery throughout the brain and spinal cord, particularly to neuronal populations. Both transduce the adult mouse brain >10-fold more efficiently than the clinical gold standard AAV9 upon intravascular infusion, with gene transfer to multiple neuronal sub-populations. These vectors are also capable of neuronal transduction in a normal cat. We have demonstrated the efficacy of AAV-AS in the context of Huntington's disease by knocking down huntingtin mRNA 33-50% after a single intravenous injection, which is better than what can be achieved by AAV9 at the particular dose. AAVB1 additionally transduces muscle, beta cells, pulmonary alveoli and retinal vasculature at high efficiency, and has reduced sensitivity to neutralizing antibodies in human sera. Generation of this vector toolbox represents a major step towards gaining genetic access to the entire CNS, and provides a platform to develop new gene therapies for neurodegenerative disorders.

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Steines, Benjamin Richard. « Investigation and application of novel adeno-associated viral vectors for cystic fibrosis gene therapy ». Diss., University of Iowa, 2015. https://ir.uiowa.edu/etd/1763.

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Cystic Fibrosis (CF) is a lethal autosomal recessive genetic disorder caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR transports anions at the apical surface of epithelial membranes and functions in many areas of the body. However in CF, loss of CFTR function in the lungs is the major source of morbidity and mortality. Replacing the defective CFTR in the lungs through gene therapy has the potential to cure the disease. Recombinant adeno-associated virus (AAV) is an effective gene transfer vector and has been used extensively to deliver genes to cells in culture. A number of clinical trials using AAV have been attempted for a variety of diseases, including CF, albeit with limited success. Poor vector transduction efficiency prevents effective gene therapy. We have previously used a technique to greatly increase the transduction efficiency of AAV in human lung tissues by selecting from a library of AAVs using a directed evolution technique. However, this evolution was performed in cultured cells and did not fully represent the in vivo environment in which the AAV would be used. In 2008, a CF pig model was developed to develop a further understanding of the mechanisms of CF and CFTR function. We hypothesized that we could use directed evolution to select for a vector in vivo using the pig, allowing gene therapy studies to be conducted in a physiologically relevant model of CF. We selected a novel AAV variant, called AAV2H22, which is closely related to AAV2 but with greatly increased transduction efficiency in pig airway epithelia. AAV2H22 displayed specific tropism for pig airway epithelia and saturated cell surface receptors, indicating specific binding in those cells. We found that AAV2H22-mediated gene transfer corrected chloride and bicarbonate transport defects both in vitro and in vivo. Importantly, bicarbonate transport was sufficient to normalize pH in the airway surface liquid, resulting in increased bacterial killing likely due to increased activity of antimicrobial peptides. To investigate the mechanics of the increased transduction of AAV2H22, capsid mutants were assayed for transduction efficiency. Two of the five amino acid differences between AAV2 and AAV2H22 lie at the surface and are predicted to alter capsid binding. This is consistent with the results showing specific binding in cultured airway epithelia. This research has important implications for gene therapy and investigations using AAV2H22 will increase our understanding of the biology needed to successfully treat CF.

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Ibraheim, Raed R. « Genome Engineering Goes Viral : Repurposing of Adeno-associated Viral Vectors for CRISPR-mediated in Vivo Genome Engineering ». eScholarship@UMMS, 2020. https://escholarship.umassmed.edu/gsbs_diss/1114.

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One of the major challenges facing medicine and drug discovery is the large number of genetic diseases caused by inherited mutations leading to a toxic gain-of-function, or loss-of-function of the disease protein. Microbiology offered a new glimpse of hope to address those disorders with the adaptation of the bacterial CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) defense system as a genome editing tool. Cas9 is a unique CRISPR-associated endonuclease protein that can be easily programmed with an RNA [a single-guide RNA (sgRNA)] that is complementary to nearly any DNA locus. Cas9 creates a double-stranded break (DSB) that can be exploited to knock out toxic genes or replenish therapeutic expression levels of essential proteins. In addition to a matching sgRNA sequence, Cas9 requires the presence of a short signature sequence [a protospacer adjacent motif (PAM)] flanking the target locus. Over the past few years, several Cas9-based therapeutic platforms have emerged to correct DNA mutations in a wide range of mammalian cell lines, ex vivo, and in vivo by adapting recombinant adeno-associated virus (rAAV). However, most of the applications of Cas9 in the field have been limited to Streptococcus pyogenes (SpyCas9), which, in its wild-type form, suffers from inaccurate editing at off-target sites. It is also difficult to deliver via an all-in-one (sgRNA+Cas9) rAAV approach due to its large size. In this thesis, I describe other Cas9 nucleases and their development as new AAV-based genome editing platforms for therapeutic editing in vivo in mouse disease models. In the first part of this thesis, I develop the all-in-one AAV strategy to deliver a Neisseria meningitidis Cas9 ortholog (Nme1Cas9) in mice to reduce the level of circulating cholesterol in blood. I also help characterize an enhanced Cas9 from another meningococcus strain (Nme2Cas9) and show that it is effective in performing editing not only in mammalian cell culture, but also in vivo by all-in-one AAV delivery. Additionally, I describe two AAV platforms that enable advanced editing modalities in vivo: 1) segmental DNA deletion by delivering two sgRNAs (along with Nme2Cas9) in one AAV, and 2) precise HDR-based repair by fitting Nme2Cas9, sgRNA and donor DNA within a single AAV capsid. Using these tools, we successfully treat two genetic disorders in mice, underscoring the importance of this powerful duo of AAV and Cas9 in gene therapy to advance novel treatment. Finally, I present preliminary data on how to use these AAV.Nme2Cas9 vectors to treat Alexander Disease, a rare progressive neurological disorder. These findings provide a platform for future application of gene editing in therapeutics.

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Moimas, 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.

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2007/2008
The 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.
XX Ciclo
1978

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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.

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Xu, Dan. « Cellular Immunity in Recombinant Adeno-Associated Virus Vector Mediated Gene Therapy ». The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1313504203.

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Carty, Nikisha Christine. « Recombinant AAV Gene Therapy and Delivery ». Scholar Commons, 2009. https://scholarcommons.usf.edu/etd/1890.

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Alzheimer's disease (AD), first characterized in the early 20th century, is a common form of dementia which can occur as a result of genetic mutations in the genes encoding presenilin 1, presenilin 2, or amyloid precursor protein (APP). These genetic alterations can accelerate the pathological characteristics of AD, including the formation of extracellular neuritic plaques composed of amyloid beta peptides and the formation of intracellular neurofibrillary tangles consisting of hyperphosphorylated tau protein. Ultimately, AD results in gross neuron loss in the brain which is evidenced clinically as a progressive decline in mental capacity. A strong body of scientific evidence has previously demonstrated that the driving factor in the pathogenesis of AD is potentially the accumulation of Aß peptides in the brain. Thus, reduction of Aß deposition is a major therapeutic strategy in the treatment of AD. Recently it has been suggested that Aß accumulation in the brain is modulated, not only by Aß production, but also by its degradation. Several important studies have demonstrated that Aß degradation is modulated by several endogenous zinc metalloproteases shown to have amyloid degrading capabilities. These endogenous proteases include neprilysin (NEP), endothelin converting enzyme (ECE), insulin degrading enzyme (IDE) and matrix metalloprotease 9 (MMP9). In this investigation we study the effects of upregulating expression of several of these proteases through administration of recombinant adeno-associated viral vector (rAAV) containing both endogenous and synthetic genes for ECE and NEP on amyloid deposition in amyloid precursor protein (APP) plus presenilin-1 (PS1) transgenic mice. rAAV administration directly into the brain resulted in increased expression of ECE and NEP and a substantial decrease in amyloid pathology. We were able to significantly increase the area of viral distribution by using novel delivery methods resulting in increased gene expression and distribution. These data support great potential of gene therapy as a method of treatment for neurological diseases. Optimization of gene transfer methods aimed at a particular cell type and brain region in the CNS can be accomplished using AAV serotype specificity and novel delivery techniques leading to successful gene transduction thus providing a promising therapeutic avenue through which to treat AD.

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Pacouret, Simon. « Thermostability of Adeno-Associated Virus (AAV) Vectors ». Thesis, Nantes, 2018. http://www.theses.fr/2018NANT1041/document.

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Les virus adéno-associés (AAVs) sont des virus à ADN simple brin, nonenveloppés, considérés comme des candidats de choix pour la thérapie génique. Pour augmenter les chances de succès des thérapies géniques basées sur l’AAV, des efforts sont actuellement faits pour développer de nouvelles capsides virales, qui seraient plus résistantes à l’immunité préexistante, plus spécifiques de certains tissus, et compatibles avec une production à grande échelle. L’un des défis posés par le développement de nouveaux vecteurs consiste à comprendre comment conférer de nouvelles fonctions biologiques aux capsides d’AAVs, sans compromettre leur intégrité structurale. Pour ce faire, il est nécessaire d’améliorer notre compréhension des mécanismes gouvernant la métastabilité des capsides d’AAVs. L’objectif de cette thèse était d’étudier la thermostabilité des AAVs, ses liens avec leurs propriétés biologiques, ainsi que ses applications dans le domaine du contrôle qualité des préparations d’AAVs recombinants Dans un premier temps, nous étendons les travaux existants à l’étude de virus AAVs ancestraux (AncAAVs), reconstruits in silico. Nous montrons que Anc80, l’ancêtre commun prédit d’AAV1, 2, 8 et 9, est plus thermostable que ses descendants (ΔT = 15-20°C). Nous identifions ensuite, par une analyse de type phénotype-phylogénie, 12 acides aminés jouant potentiellement un rôle important dans la stabilisation des capsides virales. Nous montrons ensuite que la thermostabilité des capsides d’AAVs, mesurée par fluorimétrie différentielle à balayage (DSF), est utile pour déterminer, à l’échelle protéique, l’identité des préparations de vecteurs viraux, une opération requise par les agences réglementaires. Pour finir, nous appliquons ce test d’identité à l’étude de l’homogénéité structurale des librairies d’AAVs. Ces travaux de thèse pourraient s’avérer utiles pour développement et le manufacturing de nouveaux AAVs recombinants pour la thérapie génique
Adeno-associated virus (AAV) vectors have emerged as promising gene delivery vehicles for gene therapy. To improve the probability of success of AAV-based therapeutic strategies, efforts are currently being made to engineer novel capsids able to produce and purify well, escape pre-existing immunity, and target specific cell populations more efficiently. One challenge in AAV vector engineering is to understand how to confer new functions to the viral capsid without altering its structural integrity. To do so, there is a critical need to gain further knowledge on the mechanisms steering AAV capsid metastability. The objective of this thesis is to investigate the thermal stability of AAVs, its impact on AAV biology, and applications to quality control of AAV preparations. First, we extend existing thermal stability studies to in silico reconstructed ancestral AAV particles (AncAAVs), and show that, Anc80, the common putative ancestor of AAV1, 2, 8 and 9, is 15-20°C more thermostable than its contemporary homologs. Using phenotype-tophylogeny mapping, we also identify a set of 12 residues potentially playing a key role in capsid metastability. Second, we demonstrate that capsid thermal stability, as measured by Differential Scanning Fluorimetry (DSF), can be used for identification of AAV preparations at the protein level, a requirement of regulatory agencies. Last, we apply this identity assay to the study of capsid mosaic formation in AAV library preparations. This work will help guide the engineering and manufacturing of improved AAV vectors for gene therapy

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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.

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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.

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Stachler, Matthew D. « Design and engineering of capsid modified AAV-Based vectors targeted towards angiogenic and proliferating vasculature ». The Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=osu1180370373.

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Ghosh, 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.

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Thesis (Ph. D.)--University of Missouri-Columbia, 2007.
The 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.

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Best, Victoria Maria. « Ongoing cellular responses to transgene products encoded by recombinant adeno-associated virus (rAAV) vectors ». The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1262213552.

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Koo, Taeyoung. « Studies on gene transfer in skeletal muscle cells and tissues using recombinant adeno-associated virus (AAV) vectors ». Thesis, Royal Holloway, University of London, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.529039.

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Peden, 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.

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Thesis (Ph.D.)--University of Florida, 2004.
Typescript. Title from title page of source document. Document formatted into pages; contains 134 pages. Includes Vita. Includes bibliographical references.

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Ekstedt, Elias, Inna Fryckstedt, Hanna Hyllander, Josefin Jonsson, Elin Ring et 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.

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Gene therapy is a fast growing technology that offers treatments for genetic diseases. The method is based on introducing genetic material into a patient to replace the disease-causing gene, using a vector. This report examines the potential of some viral vectors for gene therapy, to give Bio-Works Technologies a recommendation on what the future market demands. Oncolytic viruses, vaccines and gene editing are not treated in the report as a delimitation. Viral vectors have different biological properties and require different purification methods, making them suitable for different applications in gene therapy. In the purification of the viruses it can be challenging to obtain a high purity and large-scale manufacturing. One major drawback with most purification methods is that they are not specific to just one virus, which leads to contaminants in the solution and lower purity. The viral vectors handled in the report are the adenovirus, adeno-associated virus, gammaretrovirus, lentivirus, alpharetrovirus, foamy virus, herpes simplex virus and baculovirus. These were chosen as they are relevant vectors for gene therapy and stay within the scope of the report. Lentiviral vectors (LVs) and adeno-associated viral vectors (AAVs) will dominate the gene therapy field in the coming years. This is based on the information that the use of AAVs and LVs in clinical trials have increased in recent years, while the other vectors mentioned above have slightly decreased or show no apparent change. However, challenges still remain in the purification processes. Ligands used in affinity chromatography for purification of AAVs are effective at removing most contaminants, but cannot distinguish between empty and loaded capsids, which can induce immune response when used clinically. This is the main challenge when purifying AAVs. The empty capsids can be removed with ion exchange chromatography, which results in higher purity but also lower recovery. There is no specific purifying method for LVs, therefore a lentivirus-specific affinity ligand, such as an antibody ligand, would be beneficial for the purification and manufacturing procedure. In addition to AAVs and LVs, baculoviral vectors and foamy viral vectors show great potential in a long-term perspective but they only have been researched in preclinical studies. Moreover, herpes simplex viral vectors and adenoviral vectors show potential in cancer treatments or as vaccines rather than in augmentation gene therapy.

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Rossi, Axel. « Intracellular fate of AAV particles in human Dendritic Cell and impact on Gene Transfer ». Thesis, Lyon, 2016. http://www.theses.fr/2016LYSEN028.

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Les vecteurs viraux dérivés du virus adéno-associé (AAV) apparaissent depuis deux décennies, comme des outils efficaces pour le transfert de gène in vivo. Cependant, malgré une faible immunogénicité et une absence de toxicité in vivo, leur optimisation requiert encore un effort important vers une meilleure compréhension de leur biologie et, en particulier, de leur interaction avec le système immunitaire. Au cours de ce travail de thèse, nous avons utilisé une méthode de sélection dirigée in vitro dans le but d’obtenir un variant de capside capable de transduire efficacement un type cellulaire non-permissif aux vecteurs AAV : les cellules dendritiques (DC). En effet, ces cellules jouent un rôle primordial dans l’établissement de la réponse immunitaire et, par conséquent, dans la persistance de l’expression du transgène in vivo. Cette technologie, très répandue dans la communauté AAV, a permis de sélectionner un variant de capside aux propriétés très intéressantes. La mutation sélectionnée, caractérisée in vitro comme induisant une instabilité de la capside, a permis d’identifier et de surmonter un point de blocage majeur dans le processus de transduction des DC par les vecteurs AAV consistant dans l’étape de décapsidation du génome du vecteur dans le noyau cellulaire. De manière intéressante, le variant obtenu exhibe un avantage en terme de transduction non seulement dans les DC mais aussi dans différents modèles de cellules primaires humaines (e.g. HUVEC) ou animales (OBC), peu ou pas permissive à l’AAV. De plus, des expériences de transfert de gène in vivo réalisées dans un modèle murin, indiquent que le variant sélectionné conduit à une meilleure expression du transgène, possiblement due à la mise en place d’un processus de tolérisation. Les propriétés remarquables de ce variant de capside, font de lui un candidat intéressant pour des applications médicales
Vectors derived from the Adeno-associated virus (AAV) have emerged as an efficient system for in vivo gene transfer. However, despite their low immunogenicity and good tolerance in vivo, a better characterization of the host-AAV interaction is required to be able to fully exploit AAV’s potential fora gene therapy or gene vaccination. In this PhD project, we have used an in vitro directed evolution strategy to select an AAV capsid variant able to transduce human dendritic cell (DC), a non-permissive cell type which plays a critical role in the initiation of immune responses and, consequently, on the persistence of the expression of transgene in vivo. This procedure allowed us to identify an AAV variant characterized by a decreased stability of the capsid in vitro. The use of this mutant as a vector to transduce human DC resulted in an improved uncoating of the vector genome in the cell nucleus, thus identifying this step as major barrier toward DC transduction. Interestingly, the selected variant also displayed an increased transduction efficiency not only in DC but also in different primary human and animal cell types, poorly or non-permissive to AAV. Finally, when injected in mice, this AAV variant resulted in a higher expression of the transgene, associated to a low level of immune responses, suggesting the induction of tolerant state. The remarkable features suggest that our selected variant capsid is a promising candidate for medical applications

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Rohwedder, Carolin [Verfasser], et 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.

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Ippodrino, Rudy. « High-throughput RNA interference screening identifies human genes regulating AAV transduction ». Doctoral thesis, Scuola Normale Superiore, 2015. http://hdl.handle.net/11384/85970.

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Oliveira, Mónica Catarina Castro. « Proteasome-proteins : are these putative targets for basal-like breast cancer therapy with AAV-vectors ? » Master's thesis, Universidade de Aveiro, 2016. http://hdl.handle.net/10773/18553.

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Mestrado em Biologia Molecular e Celular
O cancro da mama do tipo basal (BLBC) é um grupo de tumores muito agressivo associado a um mau prognóstico. De momento, não existe nenhum tratamento eficaz para o BLBC, uma vez que rapidamente adquirem resistência às terapias normalmente usadas. Assim, é urgente encontrar novas abordagens para tratar esta doença. Com base em dados anteriores, o objetivo geral deste estudo foi avaliar se o PSMA2, uma proteína do proteassoma, seria um alvo putativo para a inibição para terapia em BLBC. Desta forma, o primeiro objetivo específico foi avaliar o efeito anti-tumorigénico de vírus adeno-associados (AAV) capazes de entregar short hairpin RNAs (shRNA), anteriormente validados, capazes de inibir a expressão do PSMA2 em xenotransplantes de células BLBC em ratinho. Para atingir esse objetivo, foram testados in vivo, vetores AAV2 com shRNAs para os genes PLK1 e PSMA2 para diferentes concentrações de partículas virais (2x1010, 2x109, 2x108 partículas virais/tumor), em que células MDA-MB-468 BLBC foram injetadas na mama de ratinhos nude. Após cerca de um mês, foram realizadas injeções intratumorais com AAVs duas vezes por semana. A administração de AAV2-shPSMA2 resultou numa diminuição no crescimento do tumor sem toxicidade evidente, e este efeito foi mais significativo na concentração de 2x109 partículas virais/tumor. O segundo objetivo específico foi analisar a expressão de PSMA2 em amostras humanas de cancro da mama, o que indica que há também uma importância clínica na inibição deste gene, uma vez que se mostrou estar associado a características menos favoráveis relacionadas com tumores da mama do tipo basal. Em conclusão, embora ainda preliminar, os resultados obtidos abrem a possibilidade de direcionar uma terapia genética em BLBC usando vetores AAV recombinantes que entregam shRNAs para silenciar especificamente a expressão do gene PSMA2.
Basal-like breast cancer (BLBC) is an aggressive group of tumours associated to poor patient prognosis. Currently, there is no effective treatment for BLBC once they rapidly acquire resistance to standard therapies. For this reason, novel approaches to treat this disease are urgently needed. Based on previous data, the general goal of this study was to evaluate if PSMA2, a proteasome protein, was a putative target for inhibition in BLBC therapy. In this way, the first specific aim was to evaluate the anti-tumorigenic effect of adeno-associated virus (AAV)-based vectors, that were able to deliver validated short hairpin RNAs (shRNAs) that inhibit the expression of PSMA2 in BLBC mouse xenografts. To achieve that aim, we have tested, in vivo, AAV2 vectors with shRNAs for the genes PLK1 and PSMA2 for different concentrations of viral particles (2x1010, 2x109, 2x108 VP/tumour), MDA-MB-468 BLBC cells were injected into the mammary fat pad of nude mice and, after nearly one month, intratumoral injections with AAVs were performed twice a week. The delivery of AAV2-shPSMA2 resulted in a decrease in tumour growth with no obvious toxicity, and this effect was more significant at the concentration of 2x109 VP/mouse. The second specific aim was to analyse the expression of PSMA2 in human breast cancer samples, which indicated that there is also a clinical importance in inhibiting this gene, once it showed to be associated with less favourable features that are linked to basal-like breast tumours. In conclusion, although still preliminary, the results obtained open a possibility to direct a gene-based therapy in BLBC using recombinant AAVs that deliver shRNAs that specifically silence PSMA2 gene expression.

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Schnödt, Maria Angelika [Verfasser], Dagmar [Gutachter] Mörsdorf, Mirka [Gutachter] Uhlirova et 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.

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Cataldi, 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.

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Weinmann, Jonas [Verfasser], et 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.

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Sallach, Jessica [Verfasser], Dagmar [Akademischer Betreuer] Knebel-Mörsdorf et 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.

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Pagès, 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.

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La mucopolisacaridosi tipus VII (MPS VII) és una malaltia monogènica molt rara inclosa en el grup de malalties lisosòmiques. Està causada per la manca d’activitat β-‐ glucuronidasa, un enzim lisosòmic involucrat en la via de degradació de glicosaminoglicans. Aquesta alteració congènita causa una disfunció del sistema lisosòmic que provoca una acumulació anormal als lisosomes i la disrupció de l’homeòstasi de la cèl·lula. La malaltia presenta diferents graus de severitat clínica que van des de la mort prenatal fins a pacients amb formes lleus i una esperança de vida de 20 o 30 anys. Entre els casos que presenten un inici postnatal de la malaltia, la forma més severa de MPS VII es caracteritza per hepatomegàlia, esplenomegàlia, anomalies esquelètiques, desenvolupament tardà i retard mental, entre altres símptomes. Actualment els tractaments per als pacients de MPS VII són intervencions per pal·liar els símptomes de la malaltia, però no disposen de cap tractament curatiu. La teràpia gènica és una estratègia prometedora de cara a trobar una cura per a malalties monogèniques. Entre els vectors de teràpia gènica disponibles, els virus adeno-‐associats (AAV) presenten certes característiques que els fan molt atractius per a la teràpia gènica: permeten la transducció tant de cèl·lules en divisió com quiescents, proporcionen una expressió del transgèn a llarg termini, no poden replicar-‐se de manera autònoma sense un virus accessori i, a més, les infeccions naturals dels AAV no són patogèniques. Diferents serotips d’AAV s’han utilitzat com a vectors de teràpia gènica en estudis preclínics. Entre ells, els serotips AAV9 i AAVrh10 són els que presenten una capacitat de transducció més gran, així com una gamma més àmplia de tipus cel·lulars que poden transduir, especialment al sistema nerviós central. El serotip AAVrh10 no és d’origen humà. Per això, el fet d’utilitzar-‐lo com a vector de teràpia gènica podria evitar la neutralització per part dels factors immunològics específics contra AAV, factors que són presents en el sèrum humà després d’infeccions d’AAV naturals. Tanmateix, el reconeixement creuat per part d’anticossos generats contra AAV2, el serotip humà més comú, podrien interferir en el resultat de la teràpia. En aquest treball es proposa una estratègia de teràpia gènica per a MPS VII basada en una única injecció intratecal d’un vector AAVrh10 que conté el gen de la β-‐ glucuronidasa, testada en ratolins MPS VII adults joves. Es mostra que l’administració del vector al líquid cefaloraquidi (LCR) per punció lumbar, una tècnica molt poc invasiva, permet la transducció d’estructures del sistema nerviós central (SNC) utilitzant una dosi de vector més baixa que mitjançant injecció intravenosa. A més, el drenatge del vector del LCR cap al torrent sanguini comporta la transducció d’òrgans somàtics com el fetge. Això genera una font de β-‐glucuronidasa que aconsegueix una activitat enzimàtica en sèrum comparable a la de ratolins sans. L’expressió sostinguda de l’enzim recombinant per part de les cèl·lules transduïdes, així com la correcció creuada deguda a la secreció de l’enzim, aconsegueixen la correcció de les característiques bioquímiques i histopatològiques de la malaltia, tant al SNC com als òrgans somàtics. Aquesta correcció a nivell cel·lular condueix a una millora significativa de les capacitats físiques, cognitives i emocionals dels ratolins MPS VII i aconsegueix doblar la seva esperança de vida.
Mucopolysaccharidosis 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.

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Guhasarkar, Dwijit. « A Walk on the Fine Line Between Reward and Risk : AAV-IFNβ Gene Therapy for Glioblastoma : A Dissertation ». eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/843.

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Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. The current standard-of-care treatment including surgery, radiation and temozolomide (TMZ) chemotherapy does not prolong the survival satisfactorily. Here we have tested the feasibility, efficacy and safety of a potential gene therapy approach using AAV as gene delivery vehicle for treatment of GBM. Interferon-beta (IFNβ) is a cytokine molecule also having pleiotropic anticancerous properties. Previously it has been shown by our group that AAV mediated local (intracranial) gene delivery of human IFNβ (hIFNβ) could be an effective treatment for non-invasive human glioblastoma (U87) in orthotopic xenograft mouse model.But as one of the major challenges to treat GBM effectively in clinics is its highly invasive property, in the current study we first sought to test the efficacy of our therapeutic model in a highly invasive human GBM (GBM8) xenograft mouse model. One major limitation of using the xenograft mouse model is that these mice are immune-compromised. Moreover, as IFNβ does not interact with cross-species receptors, the influence of immune systems on GBM remains largely untested. Therefore to test the therapeutic approach in an immune-competent mouse model, we next treated a syngeneic mouse GBM model (GL261) in an immune-competent mouse (C57B6) with the gene encoding the species-matched IFNβ (mIFNβ). We also tested if combination of this IFNβ gene therapy with the current standard chemotherapeutic drug (TMZ) is more effective than any one of the therapeutic modes alone. Finally, we tested the long term safety of the AAV-mIFNβ local gene therapy in healthy C57B6 mice. Next, we hypothesized that global genetic engineering of brain cells expressing secretory therapeutic protein like hIFNβ could be more beneficial for treatment of invasive, migratory and distal multifocal GBM. We tested this hypothesis using systemic delivery of AAV9 vectors encoding hIFNβ gene for treatment of GBM8 tumor in nude mice. Using in vivo bioluminescence imaging of tumor associated firefly luciferase activity, long term survival assay and histological analysis of the brains we have shown that local treatment of AAV-hIFNβ for highly invasive human GBM8 is therapeutically beneficial at an early growth phase of tumor. However, systemic delivery route treatment is far superior for treating multifocal distal GBM8 tumors. Nonetheless, for both delivery routes, treatment efficacy is significantly reduced when treated at a later growth phase of the tumor. In syngeneic GL261 tumor model study, we show that local AAV-mIFNβ gene therapy alone or in combination with TMZ treatment can provide significant survival benefit over control or only TMZ treatment, respectively. However, the animals eventually succumb to the tumor. Safety study in the healthy animals shows significant body weight loss in some treatment groups, whereas one group shows long term survival without any weight loss or any noticeable changes in the external appearances. However, histological analysis indicates marked demyelinating neurotoxic effects upon long term exposures to mIFNβ over-expressions in brain. Overall, we conclude from this study that AAV-IFNβ gene therapy has great therapeutic potential for GBM treatment in future, but the therapeutic window is small and long term continuous expression could have severe deleterious effects on health.

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Guhasarkar, Dwijit. « A Walk on the Fine Line Between Reward and Risk : AAV-IFNβ Gene Therapy for Glioblastoma : A Dissertation ». eScholarship@UMMS, 2007. http://escholarship.umassmed.edu/gsbs_diss/843.

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Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. The current standard-of-care treatment including surgery, radiation and temozolomide (TMZ) chemotherapy does not prolong the survival satisfactorily. Here we have tested the feasibility, efficacy and safety of a potential gene therapy approach using AAV as gene delivery vehicle for treatment of GBM. Interferon-beta (IFNβ) is a cytokine molecule also having pleiotropic anticancerous properties. Previously it has been shown by our group that AAV mediated local (intracranial) gene delivery of human IFNβ (hIFNβ) could be an effective treatment for non-invasive human glioblastoma (U87) in orthotopic xenograft mouse model.But as one of the major challenges to treat GBM effectively in clinics is its highly invasive property, in the current study we first sought to test the efficacy of our therapeutic model in a highly invasive human GBM (GBM8) xenograft mouse model. One major limitation of using the xenograft mouse model is that these mice are immune-compromised. Moreover, as IFNβ does not interact with cross-species receptors, the influence of immune systems on GBM remains largely untested. Therefore to test the therapeutic approach in an immune-competent mouse model, we next treated a syngeneic mouse GBM model (GL261) in an immune-competent mouse (C57B6) with the gene encoding the species-matched IFNβ (mIFNβ). We also tested if combination of this IFNβ gene therapy with the current standard chemotherapeutic drug (TMZ) is more effective than any one of the therapeutic modes alone. Finally, we tested the long term safety of the AAV-mIFNβ local gene therapy in healthy C57B6 mice. Next, we hypothesized that global genetic engineering of brain cells expressing secretory therapeutic protein like hIFNβ could be more beneficial for treatment of invasive, migratory and distal multifocal GBM. We tested this hypothesis using systemic delivery of AAV9 vectors encoding hIFNβ gene for treatment of GBM8 tumor in nude mice. Using in vivo bioluminescence imaging of tumor associated firefly luciferase activity, long term survival assay and histological analysis of the brains we have shown that local treatment of AAV-hIFNβ for highly invasive human GBM8 is therapeutically beneficial at an early growth phase of tumor. However, systemic delivery route treatment is far superior for treating multifocal distal GBM8 tumors. Nonetheless, for both delivery routes, treatment efficacy is significantly reduced when treated at a later growth phase of the tumor. In syngeneic GL261 tumor model study, we show that local AAV-mIFNβ gene therapy alone or in combination with TMZ treatment can provide significant survival benefit over control or only TMZ treatment, respectively. However, the animals eventually succumb to the tumor. Safety study in the healthy animals shows significant body weight loss in some treatment groups, whereas one group shows long term survival without any weight loss or any noticeable changes in the external appearances. However, histological analysis indicates marked demyelinating neurotoxic effects upon long term exposures to mIFNβ over-expressions in brain. Overall, we conclude from this study that AAV-IFNβ gene therapy has great therapeutic potential for GBM treatment in future, but the therapeutic window is small and long term continuous expression could have severe deleterious effects on health.

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Afonso, 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.

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Os vírus adeno-associados (AAV) têm 25 nm de diâmetro e transportam uma molécula de DNA de cadeia simples. O seu genoma está flanqueado por duas repetições terminais invertidas e codifica para proteínas de replicação, proteínas da cápside e para a proteína AAP. É um vírus cuja replicação depende da co-infeção com outro vírus, como por exemplo o adenovírus ou o herpes simplex vírus, pois não possui a capacidade de se replicar por si só. Até hoje foram descritos 13 serótipos de AAV e não existe na literatura doenças associadas à sua infeção. Os vetores AAV demonstraram ser sistemas de entrega seguros e fiáveis para entrega de transgenes terapêuticos no sistema nervoso central, mas também noutros órgãos e tecidos. O sistema nervoso central possui um caráter imuno privilegiado, uma vez que, está protegido pelas meninges, pela barreira hemato-encefálica e pelo líquido cefalorraquidiano, que em conjunto previnem a passagem de agentes nocivos exógenos, mas também de agentes terapêuticos. É, portanto, urgente encontrar novos métodos para que o transgene terapêutico atinja as células alvo no tratamento ou na cura de doenças neurológicas. O presente estudo pretende investigar as propriedades do novo vetor mosaico AAV1/AAV2 injetado no estriado e nos núcleos profundos do cerebelo (DCN), comparando-o com os serótipos parentais AAV1 e AAV2. Os critérios avaliados foram a biodistribuição do vetor pelo cérebro, as regiões do cérebro que é capaz de transduzir, o tipo de células que infeta e a toxicidade inerente à sua administração. O novo vetor mosaico AAV1/AAV2 é um AAV recombinante que transporta uma cadeia de DNA simples constituído por as repetições terminais repetidas do serótipo AAV2, o gene que codifica para a proteína green flourescent protein (GFP) e pelo promotor do citomegalovírus. A particularidade que diferencia este vetor dos já existentes, é a constituição da sua cápside, uma vez que a mesma é constituída por proteínas da cápside do serótipo AAV1 e do serótipo AAV2, originando-se por isso uma cápside tipo mosaico, daí o nome, vetor mosaico AAV1/AAV2. Ao possuir estes dois serótipos como constituintes da sua cápside, é possível que o vetor mosaico contenha características das cápside dos vetores parentais tais como, a capacidade de transduzir as células neuronais, característica dos AAV1 e ao mesmo tempo seja capaz de reconhecer o recetor de heparina que é comumente conhecido por ser o recetor primário do AAV2. Este recetor é por isso utilizado para o processo de purificação dos vetores recombinantes do AAV2, através de cromatografia por afinidade à heparina. Desta forma, há a possibilidade do vetor mosaico AAV1/AAV2 poder ser purificado pelo mesmo método. O método da tripla transfecção foi utilizado para a produção dos vetores virais. Este consiste na transfecção de uma linha celular de packaging (HEK293) com três plasmídeos: um plasmídeo helper, um plasmídeo com o transgene de interesse e um plasmídeo que codifica para as proteínas da cápside; que, neste caso específico, são dois plasmídeos, um codificante para as proteínas da cápside do serótipo do AAV1 e outro codificante para as proteínas da cápside do serótipo AAV2. Do mesmo, resulta a produção de um novo vetor com aproximadamente 50% de proteínas da cápside do serótipo AAV1 e os restantes 50% do serótipo AAV2. Numa primeira experiência, murganhos C57BL/6 foram injetados no estriado com 3×109 genomas virais (vg) do vetor mosaico AAV1/AAV2 ou com um dos vetores dos serótipos parentais, AAV1 ou AAV2. Numa segunda experiência, os murganhos foram injetados nos núcleos profundos do cerebelo com 5×109 vg do vetor mosaico AAV1/AAV2 ou com os vetores dos serótipos parentais. Um mês após a injeção, os animais foram sacrificados e o cérebro foi separado, um hemisfério para análise molecular e outro dos hemisférios para análise histológica. Os resultados sugerem que o vetor mosaico AAV1/AAV2 tem uma melhor biodistribuição e transdução comparativamente aos vetores dos serótipos parentais, aquando da sua injeção no estriado. No estriado, os vetores parentais e o vetor mosaico AAV1/AAV2 foram capazes de se espalhar desde a região da injeção para a região mais dorsal do cérebro. No cerebelo os vetores distribuíram-se desde os núcleos profundos do cerebelo para todo o cerebelo, assim como para as regiões que o cerebelo recebe e envia projeções. No estriado, o vetor mosaico AAV1/AAV2 foi o que obteve melhores resultados de transdução e biodistribuição, sendo capaz de transduzir o córtex, o estriado, tálamo, os núcleos endopiriformes, núcleos sub-talâmicos e o globus pallidus; enquanto que, aquando a injecção nos núcleos profundos do cerebelo, o vetor do serótipo AAV1 teve melhor transdução e biodistribuição, sendo capaz de transduzir o cerebelo, o tálamo, o mesencéfalo, o núcleo central da amígdala, a medula e a ponte. Tanto no estriado, como nos núcleos produndos do cerebelo não foi possível inferir o tropismo celular dos vetores parentais e o vetor mosaico AAV1/AAV2, pois nenhum co-localizou com os marcadores utilizados, NeuN, GFAP e Iba-1 que marcam respetivamente, os neurónios, astrócitos e microglia. No entanto, é possível verificar a transdução das células de Purkinje por microscopia, uma vez que, a região onde estas se localizam se encontra a expressar GFP. Os testes toxicológicos revelaram que após a administração dos vetores parentais e o vetor mosaico AAV1/AAV2 no estriado, não houve perda de marcador neuronal. Em conclusão, o vetor mosaico AAV1/AAV2 demonstrou ser um vetor seguro, com resultados que indiciam ser um bom candidato para transdução de células do sistema nervoso central. Este foi capaz de manter o tropismo celular do serótipo parental AAV1 e, simultaneamente, ser purificado pelo método de purificação utilizado para o AAV2, i.e. a cromatografia de afinidade à heparina. Aquando da injeção do vetor mosaico AAV1/AAV2 no cerebelo, a biodistribuição parece estar diminuída em relação ao serótipo AAV1, o que levanta a questão quanto ao contributo do serótipo AAV2 para o modo de dispersão do vetor nesta região. No futuro são necessários testes complementares para determinar se a utilização do vetor mosaico AAV1/AAV2 é superior relativamente aos serótipos parentais e ainda determinar a população neuronal especifica traduzida pelo vetor de interesse. Seria muito interessante que o vetor AAV1/AAV2 fosse empacotado com um gene terapêutico para ser testado, por exemplo, num modelo animal de uma doença neurodegenerativa. Caso os resultados demonstrassem que a utilização do vetor mosaico AAV1/AAV2 fossem vantajosos em relação aos vetores conhecidos, poderia vir a ser utilizado em ensaios clínicos para o tratamento ou cura de doenças neurodegenerativas.
Adeno-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.

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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.

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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.

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Perabò, Luca [Verfasser]. « Adeno-associated virus display : in vitro evolution of AAV retargeted vectors / Luca Perabò ». 2003. http://d-nb.info/972496246/34.

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Qureshi, Wajeeha Rashid. « Adeno-associated viral vectors for tissue-specific gene delivery in vivo ». Thesis, 2017. https://hdl.handle.net/2144/23998.

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OBJECTIVE: Clinical gene therapy is increasingly becoming a favorable method for the targeting and treatment of various human diseases. One such gene-transfer method is the use of adeno-associated viruses (AAVs) as a tool for tissue-specific delivery of genes. AAV vectors are attractive vector candidates due to their low pathogenicity, biological safety, and ability to infect both dividing and quiescent cells for sustained gene expression. We sought to develop a protocol for the production, propagation, purification, and characterization of various AAV serotypes to determine their tropism and to observe their integrative and expressive abilities using different reporter genes in vitro and in vivo. METHODS: We used subcloning techniques to introduce our genes of interest into an AAV expression plasmid and recombined with two other AAV vectors required for packaging, cell-specific targeting, and integration capabilities. Serotypes AAV1/8/9 and 5 which are cross-packaged with the prototype AAV2 were used to individually target cardiac cells/skeletal muscle and neuronal cells respectively in eC57BL/6J mice. Viral particles were propagated in vitro and purified using an iodixanol gradient. Purified AAV vectors were introduced into cells in culture and injected into live mice via tail vein injection or using robotic stereotaxic surgery directly into brain opposite saline controls to determine expression capabilities in vitro and in vivo, respectively. Live mice were imaged using the IVIS bioluminescent imaging system to measure luciferase luminescent readout. Separately, sections were acquired from the brains of the mice injected with serotype AAV2/5 and imaged using live cell imaging fluorescence microscopy to demonstrate integrative ability of the vectors into neurons based on GFP reporter signal. RESULTS: A protocol for the generation of neuron-specific AAVs was developed for the successful integration of the reporter gene GFP. The reporter gene was cloned into the AAV expression cassette. Efficient transfection methods were determined along with optimal culture conditions for the propagation of the viral particles in vitro. Promoter-driven reporter gene expression was observed in serotype-targeted cell types in vitro and in vivo. Expression was limited to neuronal cells based on AAV serotype specificity and SYN1 promoter. Integration and expression of the luciferase gene was not observed in the IVIS system. CONCLUSION: Here we demonstrate expression of a GFP reporter in specific neuronal cell types which indicates successful integration and expression abilities of AAV vectors. This specific tissue-targeting technique using the AAV vector highlights the potential for the further development of these vectors as a promising gene transfer system.

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Shevtsova, 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.

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Carreiras, 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.

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Dissertação de Mestrado em Biotecnologia Farmacêutica apresentada à Faculdade de Farmácia
Nas 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.

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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.

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Indiana University-Purdue University Indianapolis (IUPUI)
High-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.

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Burt, Daniel Robert. « Optimization of viral transduction in the central nervous system ». Thesis, 2014. https://hdl.handle.net/2144/14650.

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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

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Shevtsova, 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.

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Bourhill, 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.

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dissertation submitted to the Faculty of Health Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Medicine. Johannesburg, 2015
Chronic 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.

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Huang, Yung An, et 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.

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碩士
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.

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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.

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Dissertação de Mestrado em Investigação Biomédica apresentada à Faculdade de Medicina
A 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)

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Wang, Chi-Hsien, et 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.

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博士
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.

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