Dissertations / Theses on the topic 'Gene and Molecular Therapy'
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Chen, Ian Ying-Li. "Molecular imaging of cardiac gene therapy /." May be available electronically:, 2008. http://proquest.umi.com/login?COPT=REJTPTU1MTUmSU5UPTAmVkVSPTI=&clientId=12498.
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Lau, Cara Jean. "Gene therapy for malignant gliomas." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=18478.
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Gliomas are the most common primary brain tumours found in adults. The median survival of patients diagnosed with the most malignant form, glioblastoma multiforme (GBM), is 9-12 months and has changed little over the years despite advances in medical technology. Gene therapy may offer new solutions to treat this resistant disease. Hence, we tested three different gene therapy strategies. In our first study, we tested the efficacy of targeted therapy to correct common aberrations found in gliomas including amplification/mutation of receptor tyrosine kinases (RTK) and loss of PTEN, which result in an overactive PI3K/Akt pathway. Without PTEN, FOXO transcription factors are inactivated, and the cell becomes resistant to apoptosis and cell cycle arrest. By using an adenoviral vector (AdV) expressing an activated FOXO1 mutant (AdFOXO1;AAA), we restored apoptosis and cell cycle arrest, reduced tumour volume and prolonged survival in an intracerebral xenograft model. Secondly, we examined the therapeutic capacity of a novel replicating/non-disseminating AdV expressing the fusion protein of cytosine deaminase and uracil phosphoribosyltransferase (CU). CU can convert the non-toxic pro-drug, 5-fluorocytosine (5-FC) to the tissue diffusible chemotherapeutic drug, 5-fluorouracil (5-FU) to target dividing cells. In vitro, the replicating vectors were superior to the non-replicating vectors, but the fully replicating/disseminating vector did not perform considerably better than the replicating/non-disseminating vector suggesting that dissemination may not be advantageous. In vivo, the replicating/non-disseminating vector administered in conjunction with 5-FC prolonged survival in both an athymic and an immunocompetent mouse model. Moreover, an immune bystander effect in vivo was mediated by macrophages and T cells. Lastly, we investigated a method to harness a tool of the immune system, IFN-ß; this cytokine is known to have anti-angiogenic, anti-proliferative, and immunomoLes gliomes sont des tumeurs primaires de cerveau les plus communes retrouvées dans les adultes. La survie médiane des patients diagnostiqués avec la forme la plus maligne, le glioblastome multiforme (GBM), est de 9 à 12 mois et a peu changé au cours des années en dépit des avances en technologie médicale. La thérapie génique peut offrir de nouvelles solutions pour traiter cette maladie résistante. Durant nos travaux, nous avons examiné trois stratégies différentes de thérapie génique Dans notre première étude, nous avons examiné l'efficacité de la thérapie visée à corriger des anomalies communes retrouvées dans les gliomes, comprenant l'amplification/mutation de récepteurs de type tyrosine kinase (RTK) et la perte de PTEN, qui mènent en conséquence à une voie activée de PI3K/Akt. Sans PTEN, les facteurs de transcription FOXO sont inactivés, et la cellule devient résistante à l'arrêt du cycle cellulaire et à l'apoptose. En utilisant un vecteur adénoviral (AdV) exprimant une protéine activée du mutant FOXO1 (AdFOXO1;AAA.), nous avons reconstitué les signaux pour l'arrêt du cycle cellulaire et l'apoptose in vitro ainsi que in vivo. Deuxièmement, nous avons examiné la capacité thérapeutique d'un nouveau vecteur adénovirale qui a la capacité de se répliquer sans provoquer de lyse cellulaire et qui exprime en plus la protéine de fusion uracile phosphoribosyltransférase/cytosine déaminase (CU). La protéine CU peut convertir le promédicament non-toxique, le 5-fluorocytosine (5-FC) à la drogue chimiothérapeutique diffusible, le 5-fluorouracile (5-FU) qui a comme cible des cellules en division cellulaire. In vitro, les vecteurs à capacité de répliquation étaient meilleurs que ceux qui ne pouvaient pas se répliquer. In vivo, le vecteur en présence du 5-FC a prolongé la survie de deux modès animaux (avec et sans sytèmes immunitaires). Dans un dernier temps, nous avons étudié une méthode pour exprimer l'IF
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Katabi, Maha M. "Transcriptional targeting of suicide genes in cancer gene therapy." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0021/NQ55345.pdf.
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Wallace, Lindsay M. "Gene Therapy for Facioscapulohumeral Muscular Dystrophy." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1338315498.
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Rohatgi, Priyanka. "Engineering Protein Molecular Switches To Regulate Gene Expression with Small Molecules." Diss., Georgia Institute of Technology, 2006. http://hdl.handle.net/1853/19852.
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Small molecule dependent molecular switches that control gene expression are important tool in understanding biological cellular processes and for regulating gene therapy. Nuclear receptors are ligand activated transcription factors that have been engineered to selectively respond to synthetic ligands and used as regulators of gene expression. In this work the retinoid X receptor (RXR), has been used to develop an inducible molecular switch with a near drug like compound LG335. Three RXR variants (Q275C; I310M; F313I), (I268A; I310A; F313A; L436F), (I268V; A272V; I310M; F313S; L436M) were created via site-directed mutagenesis and a structure based approach, such that they preferentially bind to the synthetic ligand LG335 and not its natural ligand, 9-cis retinoic acid. These variants show reverse ligand specificity as designed and have an EC50 for LG335 of 80 nM, 30 nM, 180 nM, respectively. The ligand binding domains of the RXR variants were fused to a yeast transcription factor Gal4 DNA binding domain. This modified chimeric fusion protein showed reverse response element specificity as designed and recognized the Gal4 response element instead of the RXR response element. The modified RXR protein did not heterodimerize with wild type RXR or with other nuclear receptor such as retinoic acid receptor. These RXR-based molecular switches were tested in retroviral vectors using firefly luciferase and green fluorescence protein and they maintain their inducible behavior with LG335. These experiments demonstrate the orthogonality of RXR variants and their possible use in regulating gene therapy.
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Yoshida, Jun. "Molecular Neurosurgery Using Gene Therapy to Treat Malignant Glinoma." 名古屋大学医学部, 1996. http://hdl.handle.net/2237/6179.
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Thraser, Adrian James. "Molecular studies towards gene therapy for chronic granulomatous disease." Thesis, University College London (University of London), 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.307515.
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Tiwari, Swati. "Gene Therapy Approaches for Hemophagocytic Lymphohistiocytosis." University of Cincinnati / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1447690858.
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Mckiver, Bryan D. "SND1-Targeted Gene Therapy for Hepatocellular Carcinoma." VCU Scholars Compass, 2018. https://scholarscompass.vcu.edu/etd/5676.
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Staphylococcal nuclease and tudor-domain containing 1 (SND1) is an oncogene for a wide variety of cancers, including hepatocellular carcinoma (HCC). SND1 is a multifunctional protein regulating gene expression of proto-oncogenes and tumor suppressor genes, making SND1 a prime target for developing cancer therapeutics. This notion is especially attributed to HCC as most patients are diagnosed in advanced stages and the therapeutic options available for these patients are severely limited. In this study, we evaluated the therapeutic potential of a replication-defective adenovirus vector delivering SND1 shRNA (Ad.SND1sh) to human HCC cell lines, HepG3, HuH-7, and Hep3B. Adenovirus infection in HCC cells was confirmed by Western blotting and immunofluorescence. The efficacy of Ad.SND1sh to knockdown SND1 expression was confirmed via Western blot, qRT-PCR, and immunofluorescence. Ad.SND1sh did not significantly affect proliferation of the three human HCC cells but significantly inhibited their invasive and migratory capacities, as determined by wound healing and Matrigel invasion assays, respectively. As a corollary, Ad.SND1sh treatment resulted in a decrease in mesenchymal markers, such as N-cadherin, Twist, Snail, and Slug, without affecting levels of epithelial marker E-Cadherin, indicating that SND1 knockdown induces mesenchymal conversion in HCC cells. Additionally, reductions in liver cancer stem cell marker CD133 and HCC marker α-fetoprotein (AFP) were observed with SND1 knockdown. HCC cells with aberrant expression of these markers are associated with tumor initiation, recurrence, and multi-drug resistance. Our findings indicate that Ad.SND1sh may potentially be an effective therapy for advanced HCC and needs to be studied further for its clinical application.
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Perri, Sabrina R. "Exploiting the use of plasminogen kringle domains for cancer gene therapy." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=103176.
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Angiostatin is one of the most widely studied inhibitors of angiogenesis and encompasses the first 3 or 4 kringle domains of human plasminogen (Plg). Of particular interest, is the fifth kringle of human Plg (KS), which displays higher anti-angiogenic potency than angiostatin. In fact, kringles 1 to 5 exhibit greater inhibitory activity against endothelial cells than angiostatin, and this finding suggests that K5 domain acts synergistically to enhance the anti-angiogenic effect of angiostatin. Therefore, we proposed that the K5 domain---on its own---could act as a potent anti-angiogenic and anti-tumor protein within the context of a cancer gene therapy strategy.In our first study, we assessed the angiostatic properties of the K5 peptide domain in an orthotopic brain cancer model. We demonstrated that the disulfide bridging conformation of K5, necessary to maintain its functionality, is conserved upon secretion by gene-modified mammalian cells. Kringle 5 retrovirally gene-engineered human U87 glioma cells produced functional soluble K5 protein capable of suppressing growth factor-induced endothelial cell migration in vitro and inhibiting glioma-induced angiogenesis in vivo. Interestingly, secreted K5 protein blocked the recruitment of tumor-associated CD45+Mac3 +Grl- macrophages in vivo and inhibited the migration of CD206+ human monocyte-derived macrophages in vitro. Moreover, in a clinically relevant orthotopic glioma model, soluble K5 induced long-term survival in a majority of test animals. Thus, these findings validate the use of a gene therapy approach to deliver Plg K5 protein and suggest that K5 acts as a novel 2-pronged anti-tumor agent, mediating its inhibitory effect via its action on host-derived endothelial cells and tumor-associated macrophages.
To determine if K5 mediated its anti-tumor effect by modulating other immune effector cells, we tested the use of soluble K5 in a murine DA/3 mammary adenocarcinoma model. Soluble K5 produced by retrovirally gene-modified DA/3 cells led to long-term survival (over 1 year) of immunocompetent BALB/c mice however, we failed to observe tumor rejection in immunodeficient NOD-SCID and BALB/c nude mice. Further analysis revealed that K5 enhances the recruitment of tumor-infiltrating CD3+ lymphoid cells, in particular the natural killer T (NKT)-lymphocyte phenotype. Consistent with our previous findings, we demonstrated that K5 led to a significant decrease in tumor-associated microvessel length and density. Interestingly, K5 tumors were characterized by a robust neutrophilic infiltrate. This may be explained by the ability of K5 to act as a strong chemotactic agent for human neutrophils in vitro as well as its ability to promote CD64+ activation within the CD11b+ neutrophil phenotype. These findings confirm that K5 acts as a potent angiostatic agent and possesses a novel pro-inflammatory role via its ability to recruit tumor-associated neutrophils and NKT-lymphocytes, leading to a strong anti-tumor response.
Tumor-associated macrophages (TAM) are key immune effector cells implicated in promoting tumor progression and metastasis. It would thus be desirable to explore strategies to reduce TAM infiltration within the tumor microenvironment. In our first study, we demonstrated that soluble K5 protein blocks macrophage recruitment. In addition, the recent observation that angiostatin reduces macrophage infiltration in an atherosclerosis model prompted our laboratory to further explore the use of angiostatin as an anti-macrophage agent. We demonstrated that angiostatin suppresses the in vitro migration of both murine peritoneal macrophages and human monocyte-derived CD206 + macrophages. Furthermore, we showed that angiostatin led to a decrease in the gelatinolytic activity of macrophage-produced matrix metalloproteinase-9, which may explain, in part, the observed angiostatin-mediated inhibition of migration. Additionally, we detected the presence of the beta-subunit of ATP synthase on the cell-surface of macrophages. ATP synthase was previously found to be a receptor for angiostatin on the cell-surface endothelial cells. We propose that the presence of ATP synthase on the surface of macrophages may promote interaction with angiostatin and prevent migration, similar to what has been reported with endothelial cells. Our findings suggest that angiostatin holds promise as an inhibitory agent against macrophages.
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Taylor, Jennifer. "Engineering and improving a molecular switch system for gene therapy applications." Diss., Georgia Institute of Technology, 2011. http://hdl.handle.net/1853/39501.
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Molecular switch systems that activate gene expression by a small molecule are effective technologies that are widely used in applied biological research. Previously, two orthogonal ligand receptor pairs (OLRP) were developed as potential molecular switch systems by modifying nuclear receptors, ligand-activated transcription factors, to bind and activate gene expression with the synthetic ligand LG335 and not with the natural ligand 9-cis retinoic acid (9cRA). The two OLRP previously discovered were RXR variant 130 (I268A, I310A, F313A, and L436F) (also known as GR130) and the RXR variant QCIMFI (Q275C, I310M, and F313I) and (also known as GRQCIMFI).
The OLRP were further developed into molecular switches to provide controlled gene expression and potentially benefit gene therapy applications by replacing the DNA binding domain (DBD) with a Gal4 DBD, a yeast transcription factor. Both molecular switches are able to bind Gal4 RE in response to LG335 and activate expression of a luciferase or GFP reporter gene in either a two- or one-component system. When characterizing the GR130 variant in the two-component system, no activation was observed with the natural ligand 9cRA, and the variant displayed a 19±5-fold activation and a 50 nM EC50 value in the presence of LG335. When the GRQCIMFI variant was evaluated in the two-component system, activation was observed in the presence of LG335 with a 10 nM EC50 value and a 6±2-fold induction, and 9cRA induced activation only at the highest concentration. The GRQCIMFI variant was also characterized with the one-component system containing the reporter gene GFP in a transient transfection as well as through retroviral transduction, displaying green fluorescence in 30% of the cells in the presence of 10 µM LG335.
Several attempts were made to improve the molecular switch system. The VP16 activation domain was fused to GRQCIMFI in an effort to increase the fold induction; however, the addition of the VP16 created a constitutively active protein. Another approach to improve the molecular switch incorporated error-prone PCR to discover a new variant, Q275C, I310M, F313I, L455M (QCIMFILM), which displayed a 10-fold increase in sensitivity towards LG335 with a 5 nM EC50 value. Examination of the L455 position in the crystal structure of RXR revealed this residue is located outside of the ligand binding pocket on helix 12 (H12), but is able to significantly enhance receptor function. In fact, the single variant, L455M, was able to enhance receptor activation, compensate for a nonfunctional variant, as well as influence coactivator association.
The long-term goal of this research is to develop a gene regulation system that would be used in human gene therapy trials. In the process of creating this system a deeper assessment of the nuclear receptor structure and function is made, which can be used for the enhancement and development of transcriptional regulation mechanisms.
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Yogalingam, Gouri. "Molecular characterisation of feline MPS VI and evaluation of gene therapy /." Title page, contents and abstract only, 1998. http://web4.library.adelaide.edu.au/theses/09PH/09phy54.pdf.
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Clarke, Don Lucas. "Development of a Stem Cell Gene Therapy for Sanfilippo Syndrome Type B." Thesis, California State University, Los Angeles, 2017. http://pqdtopen.proquest.com/#viewpdf?dispub=10278830.
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Sanfilippo syndrome type B (Mucopolysaccharidosis type IIIB; MPS IIIB) is a lysosomal storage disorder affecting primarily the brain and is characterized by profound intellectual disability, dementia, and a lifespan of about twenty years. The cause is a mutation in the gene encoding α– N-acetylglucosaminidase (NAGLU), a lysosomal enzyme, leading to the deficiency of NAGLU and accumulation of heparan sulfate. I am investigating a stem cell gene therapy approach in a Naglu-/- mouse model. I think that iNSCs overexpressing NAGLU can engraft and reduce neural pathology in the mouse model. Here I report that NAGLU overexpressed in neural stem cells derived from induced pluripotent stem cells (iNSCs) is capable of being taken up by deficient cells. I used flow cytometry and Lysotracker to demonstrate that NAGLU can reduce deficient cells’ lysosomal volume in vitro, suggesting that NAGLU treatment has a biological effect. iNSCs overexpressing NAGLU were injected into the brains of 1 day old Naglu-/- mice. iNSCs were detected 10 weeks after injection. Brain sections possessed NAGLU activity greater than or equal to heterozygous controls, activity was detected distal to injection sites, and transplanted animals showed reduction in LAMP1, GFAP, and CD68. The results suggest that engineered iNSCs could be used to deliver enzyme and treat MPS IIIB.
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Gwyther, Jacqueline Mary. "Molecular analysis and gene therapy of X-linked severe combined immunodeficiency disease." Thesis, University College London (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312004.
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Tabebordbar, Mohammadsharif. "Improving Stem Cell-Based Therapy and Developing a Novel Gene Therapy Approach for Treating Duchenne Muscular Dystrophy (DMD)." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:26718751.
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Genetic mutations in muscle structural genes can compromise myofiber integrity, causing repeated muscle damage that ultimately exhausts muscle regenerative capacity and results in devastating degenerative conditions such as Duchenne Muscular Dystrophy (DMD), Congenital Muscular Dystrophy (CMD) and different forms of Limb Girdle Muscular Dystrophy (LGMD). Gene supplementation and autologous stem cell transplant have been put forward as promising, though still unproven, therapeutic avenues for combatting these genetic muscle diseases. Both strategies aim to compensate expression of the missing or mutated protein. For cell therapy, autologous muscle stem cells (satellite cells) from dystrophic muscles undergo in vitro expansion and gene correction and then are transplanted into diseased tissue, where they fuse with resident myofibers to deliver a functional copy of the gene. One of the major obstacles for the autologous adult stem cell transplantation is that adult satellite cells account for a very rare population in muscle and they need to be expanded in culture, while retaining their engraftment potential, to generate sufficient number of cells for gene correction and transplantation. I tackled this problem by developing a culture condition that allows engraftable mouse satellite cells to expand in culture. This study also provides evidence for the feasibility of in vitro expansion, gene correction and transplantation of dystrophic satellite cells to restore DYSTROPHIN expression in dystrophic muscle.
In gene therapy, engineered gene products are delivered directly to muscle fibers as transgenes carried by viral vectors, such as Adeno Associated Viruses (AAVs). Viral- mediated delivery of a normal copy of the mutated genes into dystrophic muscle fibers holds big promise as a therapeutic avenue for Muscular Dystrophies. However, considering the indispensible role of satellite cells in muscle regeneration, an effective and long-term therapy for genetic muscle diseases requires restoration of gene expression in both dystrophic muscle fibers and satellite cells. Conventional gene therapy approaches lack the potential for long-term restoration of the mutated gene expression in satellite cells. In order to address this limitation, this study provides the proof of concept evidence for the use of a novel gene editing approach, which allows irreversible correction of the mutations in both dystrophic skeletal muscle fibers and satellite cells.Biology, Molecular and Cellular
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Larochelle, Nancy. "Gene therapy for muscular dystrophy : evaluation of a muscle-specific promoter for adenovirus-mediated gene transfer." Thesis, McGill University, 1998. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=20231.
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Replication-defective (E1+E3 deleted) human adenovirus vectors are promising means of therapeutic gene delivery to skeletal muscle cells. Since the tropism of adenovirus is non-selective, muscle-specific expression of systemically administered vectors can only be achieved by the use of a tissue-specific promoter/enhancer that is small enough to fit the insert capacity of the vector. We have generated a replication-defective adenovirus recombinant (AV) in which the reporter gene (firefly luciferase) was driven by a truncated (1.35 kb) muscle creatine kinase (MCK) promoter/enhancer. Highly efficient and muscle-specific transgene expression was demonstrated in immunodeficient mice after local injection of AV into muscles at early age. Luciferase levels produced by AVMCKlux compared favourably to those in parallel experiments from injection of AVRSVlux in which lux expression is driven by the ubiquitously active LTR sequences of RSV. In nonmuscle tissues (brain, liver, kidney, lung), the transgene expression was extremely low even though in these tissues in situ polymerase chain reaction showed as high an infectivity of the cells by the AV as in muscle, and high levels of expression were obtained with AVRSVlux. The relatively small size, the good efficiency and the muscle specificity of the MCK promoter/enhancer would make it ideal to drive the 6.3 kb (truncated) dystrophin cDNA in first generation AV (with a limited (8 kb) insert capacity) designed for gene therapy of Duchenne muscular dystrophy.
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Domingo, i. Espín Joan. "Development and characterization of artificial viruses for gene therapy." Doctoral thesis, Universitat Autònoma de Barcelona, 2013. http://hdl.handle.net/10803/123204.
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Els riscos biològics associats a la teràpia gènica viral limiten el ple desenvolupament de vectors virals i plantegen importants problemes a la seva incorporació en assajos clínics. La teràpia gènica no viral representa una alternativa segura als virus naturals per al lliurament dirigida de gens a cèl·lules, tot i els baixos nivells d'expressió gènica obtinguts que és l’obstacle principal per la seva aplicació terapèutica. Els diferents tipus de vectors no virals que s'han desenvolupat fins ara, inclouen els basats en liposomes, dendrímers o proteïnes. Recentment, el concepte de "virus artificial” s'ha proposat per descriure nanocomplexes per al lliurament de gens que imiten les funcions virals pertinents per a la captació del gen i el tràfic intracel·lular. Entre ells, els basats en proteïnes i construïts a través de principis modulars permeten la incorporació, en un únic polipèptid, de diferents proteïnes o dominis de proteïnes amb funcions similars a virus, és a dir, la unió i la condensació a ADN, la unió al receptor, la internalització, l’escapament endosomal, la localització nuclear i l’alliberament del material transportat.
Hem desenvolupat una sèrie de vehicles proteics modulars formades per diferents dominis funcionals que són capaços d'entrar en les cèl·lules a través de la unió un receptor específic i promoure nivells importants de l'expressió gènica.
En aquesta tesi s'analitza aquest enfocament amb dos articles de revisió i es demostra amb tres treballs originals.The biological risks associated to viral gene therapy limit the full development of viral vectors and pose major concerns to their incorporation into clinical trials. Non-viral gene therapy represents a safe alternative to natural viruses for cell targeted gene delivery, although the low gene expression levels achieved by non-viral vectors are a main obstacle for their therapeutic application. Different types of non-viral vectors have been developed up to date, including those based in liposomes, dendrimers or proteins. Recently, the ‘Artificial virus’ concept has been proposed to describe nanocomplexes for gene delivery that mimic the viral functions relevant to gene uptake and intracellular trafficking. Among them, those based on proteins and constructed through modular principles allow the incorporation, in a single polypeptide, of different proteins or protein domains with virus-like functions, namely DNA binding and condensation, receptor binding, internalization, endosomal escape, nuclear targeting and uncoating. We have developed a series of protein-only modular vehicles composed by different functional domains that are able to enter cells through specific receptor binding and promotes important levels of transgene expression. In this thesis this approach is discussed with two review articles and demonstrated with three original papers.
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Guo, Hong. "Molecular therapy for peritoneal fibrosis targeting the TGF-b/Smad signaling pathway /." Click to view the E-thesis via HKUTO, 2007. http://sunzi.lib.hku.hk/HKUTO/record/B39557509.
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Guo, Hong, and 郭紅. "Molecular therapy for peritoneal fibrosis: targeting the TGF-{221}/Smad signaling pathway." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2007. http://hub.hku.hk/bib/B39557509.
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Brennecke, Johannes. "Molecular diagnostics of the bacterial response to antibiotic therapy." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/28843.
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Bacterial bloodstream infections (BSIs) are a major healthcare problem causing high mortality and economic cost. BSIs require an immediate initiation of antibiotic therapy as any delay is associated with a mortality increase. With the emergence of antimicrobial resistance, the choice of the appropriate antibiotic becomes increasingly difficult, thus creating an urgent need for new diagnostics, ideally to be done at the point of care. The current gold standard is blood culture with subsequent susceptibility testing although several molecular methods have recently entered the market. However, in many instances there is a discrepancy between the in-vitro data provided by the test and the outcome of antimicrobial therapy in-vivo because current diagnostics fail to take into account the impact of the environment in the patient such as the immune system, pharmacokinetics and pharmacodynamics or bacterial fitness. In this thesis, it was hypothesised that the measurement of the bacterial gene expression after the beginning of antibiotic therapy might be a more accurate indicator of the therapy outcome because it reflects the bacterial response under in-vivo conditions. In the first part of the thesis the expression of a set of pre-defined mRNA markers was investigated under various conditions. Experiments conducted with clinical E. coli isolates incubated in human whole blood revealed an excellent correlation between the gene expression, the treatment outcome, the antibiotic susceptibility and the genetic background for three different classes of antimicrobial drugs. The second part of the thesis describes the extraction of bacterial RNA from human whole blood specimen. The effect of different agents for the lysis of human blood cells and the impact of co-purified human RNA were analysed and a method for high yield extraction of undegraded bacterial RNA was established. The third part of the thesis investigates two methods for the sensitive measurement of the bacterial gene expression. This is relevant because the bacterial loads in BSI patients are extremely low. For genes with high gene expression levels both methods yielded reliable results but were unable to quantify the expression of the previously investigated mRNA markers due to their low copy numbers. Other approaches, especially those based on single cell measurements, might be able to overcome the problem in the future and should be explored in greater detail. Overall, the foundations for a future diagnostic test based on the measurement of the bacterial gene expression have been laid in this work. Future work should address the mRNA quantification and further evaluate the connection between gene expression and therapy outcome, e.g. in animal models. A future diagnostic test should also fulfil point-of-care requirements. This will include integrated sample preparation and quantification as well as a time-to-result in the range of a few minutes.
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Wang, Xiaoxia. "Molecular studies on the action of APOBEC3G against HIV-1 and development of an APOBEC-based anti-HIV approach." American Society for Microbiology, 2011. http://hdl.handle.net/1993/23226.
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Currently, the HIV pandemic remains a major global health challenge. In order to effectively control and cure HIV-1 infection, it is necessary to perform greater research on host-HIV interactions and develop novel preventive and therapeutic approaches. The human cytidine deaminase APOBEC3G (A3G) is the first identified host restriction factor, which can serve as an initial line of defense against HIV-1 by inducing lethal mutations on proviral DNA and disrupting viral reverse transcription and integration.
In order to better understand the action of A3G on HIV-1 replication, my study was focused on characterizing the interplay between A3G and HIV-1 reverse transcriptase (RT). The results indicated that A3G directly bound to RT, which contributed to A3G-mediated inhibition of viral reverse transcription. Overexpression of the RT-binding polypeptide A3G65-132 was able to disrupt wild-type A3G and RT interaction, consequently attenuating the anti-HIV effect of A3G on HIV replication.
While the potent antiviral activities of A3G make it an attractive candidate for gene therapy, the actions of A3G can be counteracted by HIV-1 Vif during wild-type HIV infection. In order to overcome Vif-mediated blockage and maximize the antiviral activity of A3G, this protein was fused with a virus-targeting polypeptide (R88) derived from HIV-1 Vpr, and various mutations were then introduced into R88-A3G fusion protein. Results showed that Vif binding mutants R88-A3GD128K and R88-A3GP129A exhibited very potent antiviral activity, and blocked HIV-1 replication in a CD4+ T lymphocyte cell line as well as human primary cells. In an attempt to further determine their potential against drug resistant viruses and viruses produced from latently infected cells, R88-A3GD128K was chosen and delivered by an inducible lentiviral vector system. Expression of R88-A3GD128K in actively and latently HIV-1 infected cells was shown to be able to inhibit the replication of both drug sensitive and resistant strains of HIV-1.
In conclusion, this thesis has demonstrated one of the mechanisms that how A3G can disrupt HIV-1 reverse transcription. Meanwhile, an A3G-based anti-HIV-1 strategy has been developed, which provides a proof-of-principle for a new gene therapy approach against this deadly virus.
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Heller, Kristin Noreen. "Alternative to Gene Replacement for Duchenne Muscular Dystrophy using Human Alpha7 Integrin (ITGA7)." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1388401639.
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Zadro-Lamoureux, Laura. "XIAP (X-linked Inhibitor of Apoptosis) gene therapy protects photoreceptors in an animal model of retinal detachment-induced apoptosis." Thesis, University of Ottawa (Canada), 2009. http://hdl.handle.net/10393/28142.
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Retinal detachments cause photoreceptor apoptosis. XIAP (X-linked inhibitor of apoptosis) inhibits caspases-3, -7, and -9, which prevents the apoptotic cascade. This study evaluates XIAP gene therapy as a means to provide photoreceptor neuroprotection following retinal detachment.
Subretinal injections of virally-delivered XIAP or green fluorescent protein (GFP; injection control) were performed in rats. Two weeks later, retinal detachments were created at the viral injection site. Eyes were harvested 24 hours post-detachment to analyze caspase activity and at 3 days and 2 months for histological analysis.
Caspase assays indicated rises in caspase-3 and -9 activities in detached GFP-treated retinas, whereas XIAP-treated retinas behaved comparably to attached controls. Three day TUNEL analysis showed less apoptosis in XIAP-treated detachments. Two month histology confirmed preservation of photoreceptors in XIAP-treated detachments, whereas GFP-treated detached retinas had deteriorated significantly.
The results suggest that XIAP confers structural photoreceptor neuroprotection for at least two months following retinal detachment.
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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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Gade, Terence Peter Ferrante. "Integrated imaging of drug delivery : a molecular imaging approach to the optimization of cancer therapy /." Access full-text from WCMC:, 2007. http://proquest.umi.com/pqdweb?did=1432803381&sid=12&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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Connolly, Richard J. "Plasma Mediated Molecular Delivery." Scholar Commons, 2010. http://scholarcommons.usf.edu/etd/3450.
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Non-viral delivery of plasmid DNA has traditionally relied upon physical forces applied directly to target tissues. These physical methods typically involve contact between an applicator and the target tissue and often cause transient patient discomfort. To overcome the contact-dependent limitations of such delivery methodologies, an atmospheric direct current plasma source was developed to deposit ionized gas molecules onto localized treatment sites. The deposition of charged species onto a treatment site can lead to the establishment of an electric field with strengths similar to those used for traditional electroporation. In vitro experiments proved that this technology could transiently permeabilize cell membranes and that membrane restabilization followed first order kinetics. Optimum delivery of tracer molecules to cell suspensions occurred after 10 minutes of plasma exposure and was attained without adversely effecting cell viability.
In vivo testing of the plasma discharge demonstrated the capability of this system to deliver plasmid DNA to murine skin. Initial experiments involved the injection of plasmid DNA encoding luciferase into the dermis of C57BL/6J mice and then exposing the tissue to plasma discharge for 10 mintues. Delivery by this method resulted in increased luminescence that was as much as 19-fold greater than DNA injection alone. Follow-up optimization experiments demonstrated it was possible to obtain luminescence results that were similar in magnitude to those obtained using electroporation, which under optimum conditions resulted in about a 40-fold increase in peak luminescence. Finally, optimum conditions were used to deliver a plasmid DNA encoding for the 120 kilodalton glycoprotein present on the surface of a macrophage tropic HIV. Results from this vaccination experiment indicated this method was capable of producing antigen specific humoral immune responses at similar levels as when electroporation was utilized as the delivery method.
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Ireton, Gregory C. "Structural studies of yeast and bacterial cytosine deaminase : evolution and implications for anticancer gene therapy /." Thesis, Connect to this title online; UW restricted, 2003. http://hdl.handle.net/1773/5077.
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Kim, Soo Hyun. "Gene therapy demonstrates that muscle is not a primary target for non-cell autonomous toxicity in familial ALS." Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1164829314.
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Landry, Sebastien. "Gamma-glutamyl carboxylase gene expression in cultured marrow stromal cells : significance for cell therapy of hemophilia B." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=80310.
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Gamma-glutamyl carboxylase (GGCX) is an enzyme essential for the post-translational modification of glutamic acid residues found within the gamma-carboxyglutamic acid (GLA) domain of vitamin K-dependent blood coagulation factors as well as other proteins principally involved in bone development such as Osteocalcin and Matrix Gla Protein. We propose that Marrow Stromal Cells (MSC) may serve as a useful Factor IX (FIX) delivery vehicle in vivo. As part of the validation of this cellular delivery platform for gene therapy, we determined whether MSCs endogenously express the GGCX gene. We demonstrated that GGCX was present in MSCs and that it was upregulated as MSCs differentiate into osteoblasts. These results will be of use in the rational development of Marrow Stromal Cells as a delivery vehicle of synthetic gamma-carboxylated therapeutic proteins, including FIX for therapy of Hemophilia B. Furthermore, we wanted to improve upon factor IX gamma-glutamyl carboxylation by generating a fusion FIX protein with enhance carboxylation capabilities. We interchanged the propeptide responsible of the efficiency of this post-translational process of factor IX by the one of the most efficiently gamma-carboxylated proteins, prothrombin.
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Zhang, Xin. "Evaluation of hydrophobically modified low molecular weight chitosans as novel delivery vectors for non-viral gene therapy." Université Louis Pasteur (Strasbourg) (1971-2008), 2008. http://www.theses.fr/2008STR13003.
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Le succès de la thérapie génique dépend du développement de vecteurs capables de délivrer des gènes dans les cellules. Parmi les vecteurs non viraux, les chitosans sont biocompatibles et peu toxiques. Nous avons développé des chitosans de faible masse moléculaire, greffés par des chaînes hydrophobes (HM-LMW-chs) et présentant des propriétés de détergent. HM(3%)-LMW-Ch forme avec l’ADN de petites particules positivement chargées, résistantes aux nucléases et interagissant peu avec le sérum. Ces particules sont efficacement internalisées, peu toxiques pour les cellules et après administration systémique, délivrent efficacement des gènes dans des reins de souris Par ailleurs, nous avons développé un nouveau chromophore à forte absorption biphotonique (TPA). Une fois conjugué au PEI, ce nouveau chromophore apparaît être un outil efficace pour marquer les nombreuses polyamines utilisées en transfection non-virale et pour caractériser leurs complexes avec l’ADNThe success of gene therapy depends on the development of vectors able to deliver therapeutic genes into the cells. Among the non-viral vectors, chitosans are interesting since they are biocompatible and low toxic. We developed hydrophobically modified low molecular weight chitosans (HM-LMW-chs) that exhibit surfactant-like properties. HM(3%)-LMW-ch forms with DNA small positively charged particles that are resistant to DNases and nucleases and marginally interact with serum components. Moreover, these particles are efficiently internalized in cells and low toxic. After systemic administration, DNA complexes with HM(3%)-LMW-ch efficiently deliver genes in mice kidneys. Moreover, we developed a new two-photon absorbing (TPA) chromophore for two-photon imaging and conjugated it with PEI. The chromophore appears as an adequate tool to label the numerous polyamines used in non-viral gene delivery and characterize their complexes with DNA in two-photon applications
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Packer, Davin R. "Leveraging the Extracellular Matrix to Create Novel Gene Therapies for the Congenital Muscular Dystrophies." The Ohio State University, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=osu1618248650165864.
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Eriksson, Emma. "Preclinical evaluation of immunostimulatory gene therapy for pancreatic cancer." Doctoral thesis, Uppsala universitet, Institutionen för immunologi, genetik och patologi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-330189.
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Pancreatic cancer is characterized by its desmoplastic tumor microenvironment and the infiltration of immunosuppressive cells. It is a devastating disease where most patients are diagnosed at a late stage and the treatment options are few. The development of new treatments is surly needed. One treatment option explored is the use of immunotherapy with the intent to activate the immune system and change the balance from pro-tumor to anti-tumor. This thesis presents the idea of using oncolytic adenoviruses called LOAd-viruses that are armed with immunostimulatory- and microenvironment-modulating transgenes. For effective treatment of pancreatic cancer, the virus needs to be able to be given in addition to standard therapy, the chemotherapy gemcitabine. In paper I, the immunomodulatory effect of gemcitabine was evaluated in blood from pancreatic cancer patients receiving their first 28-day cycle of treatment with infusions day 1, 8 and 15 followed by a resting period. Gemcitabine reduced the level of immunosup-pressive cells and molecules but the effect did not last throughout the resting period. On the other hand, gemcitabine did not affect the level or proliferative function of effector T cells indicating that gemcitabine could be combined with immunotherapy. The LOAd700 virus expresses a novel membrane-bound trimerized form of CD40L (TMZ-CD40L). In paper II, LOAd700 showed to be oncolytic in pancreatic cancer cell lines as well as being immunostimulatory as shown by its capacity to activate dendritic cells (DCs), myeloid cells, endothelium, and to promote expansion of antigen-specific T cells. In paper III, LOAd703 armed with both 4-1BBL and TMZ-CD40L was evaluated. LOAd703 gave a more profound effect than LOAd700 on activation of DCs and the virus was also capable of reducing factors in stellate cells connected to the desmo-plastic and immunosuppressive microenvironment. In paper IV, LOAd713 armed with TMZ-CD40L in combination with a single-chain variable fragment against IL-6R was evaluated. The virus could kill pancreatic cancer cells lines through oncolysis and could also reduce factors involved in desmoplasia in stellate cells. Most interestingly, LOAd713 could reduce the up-regulation of PD-1/PD-L1 in DCs after CD40 activation. Taken together, LOAd703 and LOAd713 seem to have interesting features with their combination of immunostimulation and microenvironment modulation. At present, LOAd703 is evaluated in a clinical trial for pancreatic cancer (NCT02705196).
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Hallen, Michael Ryan. "Commercialization of a Novel Wound Therapy and Scar Prevention Product." Case Western Reserve University School of Graduate Studies / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=case1378942204.
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Wreesmann, Volkert Boudewijn Singh Bhuvanesh. "Molecular-cytogenetic characterization of head and neck chancer identification of novel prognostic factors and gene targets for therapy /." [S.l. : Amsterdam : s.n.] ; Universiteit van Amsterdam [Host], 2004. http://dare.uva.nl/document/74975.
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PEREIRA, LARISSA M. "Clonagem, expressao, purificacao e caracterizacao estrutural da proteina ribossomal L10 humana recombinante." reponame:Repositório Institucional do IPEN, 2009. http://repositorio.ipen.br:8080/xmlui/handle/123456789/9478.
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Dissertacao (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP
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Herron, John Paul. "Immunoaffinity isolation of Btk´s signalosome, a proteomic approach to identifying interacting proteins." Thesis, Södertörn University College, School of Life Sciences, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:sh:diva-1114.
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The Signalosome is a term used to define a putative signalling complex, which assembles near the plasma membrane in response to external signals received at cell surface receptors and then migrates towards downstream effectors. It is proposed to regulate the level of intracellular Ca2+ and subsequent downstream signalling events. To date it has been defined to consist of BTK, BLNK, BCAP, VAV, PLCγ2 and PI3K1-4 in B-Cells.
This work entailed initiating a new proteomic approach to investigate the nature and extent of Bruton’s tyrosine kinase, Btk, involvement in the signalosome – inherently, the aim was to study multiple interactions of Btk with other molecules. By transfecting host cells with a Btk gene-transfer plasmid, virus particles were produced that were used to up-regulate and analyse the expression of Btk in three haematopoietic cell lines: B-cells, Pre-B-cells and a myeloid cancer cell. The construction of a new gene-transfer vector was successfully carried out by plasmid sub-cloning and it was subsequently found to effectively transfect the host cells and produce virus particles. The recombinant virus particles were employed with success in transducing three haematopoietic cell lines and with immunopurification and subsequent gel separation protein signalosome complexes were obtained ready for analysis by mass spectrometrical fingerprinting (to be carried out as a joint effort in Mount Sinai Hospital in Toronto, Canada).
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Unzueta, Elorza Ugutz. "De novo design of self-assembling protein nanoparticles towards the gene therapy of colorectal cancer." Doctoral thesis, Universitat Autònoma de Barcelona, 2013. http://hdl.handle.net/10803/125920.
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Hoy en día, el cáncer sigue siendo la segunda causa de muerte en el mundo. Por lo tanto, existe una gran necesidad de encontrar nuevas terapias que resulten más efectivas para su tratamiento. Las terapias actuales, lejos de ser efectivas, producen una gran toxicidad sistémica y ofrecen un bajo porcentaje de supervivencia a los pacientes, siendo la principal causa de muerte la aparición de focos metastáticos, especialmente en el cáncer de colon. Por lo tanto, mejorar la especificidad celular y evitar la aparición de focos metastaticos son los mayores retos a los que se enfrentan las futuras terapias contra el cáncer. En este contexto, la terapia génica aparece como una alternativa muy prometedora ya que ofrece la posibilidad de personalizar las terapias además de disminuir su toxicidad. Dado que la bioseguridad es uno de los parámetros que más preocupa en este tipo de terapias, las proteínas multifuncionales aparecen como uno de los vectores de terapia génica más prometedores no solo por su alta biocompatibilidad y su baja toxicidad, sino también por su gran plasticidad. Por otro lado, la necesidad de controlar el tamaño de las partículas generadas para conseguir una adecuada biodistribucion in vivo ha sido ampliamente descrita en la literatura.
En este trabajo, hemos explorado la posibilidad de modular el autoensamblaje de proteínas multifuncionales en nanoparticulas de un tamaño predefinido con tal de obtener el máximo potencial de su capacidad de entrega de ácidos nucleicos o drogas terapéuticas. En este contexto, hemos descrito parejas de tags arquitectónicos (de los cuales uno de ellos es una poli-histidina) que son capaces de inducir el autoensamblaje de las proteínas que las contienen en nanoparticulas con propiedades estructurales predefinidas. También hemos estudiado la interacción y el traffiking intracelular de estas nanoparticulas cuando las ponemos en contacto con células en cultivo, donde hemos visto que su internalización no resulta toxico para las células de mamíferos. Además, también hemos estudiado la estabilidad estructural que presentan estas nanoparticulas cuando se administra por vía intravenosa en modelos de ratón, mostrando que las interacciones intermoleculares que se generan durante el proceso de ensamblaje de las nanoparticulas in vitro, son suficientemente fuertes como para asegurar su estabilidad estructural in vivo.
Se ha descrito que el receptor CXCR4 es un elemento clave en la formación de focos metastaticos durante el desarrollo tumoral de diferentes tipos de cáncer incluyendo el cáncer de colon, para el cual actualmente no existe todavía ningún vehículo que reconozca de forma específica las células metastaticas. En este contexto, en este estudio hemos explorado la posibilidad de funcionalizar las nanoparticulas proteicas con ligandos que reconocen el receptor CXCR4 con tal de dirigirlas de forma específica a las células que expresan este receptor. Entre los ligandos testados, hemos visto que el péptido T22 es un ligando inusualmente eficiente para el reconocimiento selectivo e internalización en células que expresan el receptor CXCR4 y que las nanoparticulas funcionalizadas con este ligando se biodistribuyen de forma selectiva a las células CXCR4+ in vivo en modelos murinos de cáncer colorectal. Finalmente, también hemos explorado la posibilidad de usar nanoparticulas proteicas funcionalizadas como virus artificiales para la entrega especifica de ácidos nucleicos en las células diana. Este trabajo muestra como las nanoparticulas que contienen dominios de unión a ácidos nucleicos, cuando son incubados junto a un DNA externo, son capaces de generar estructuras que imitan las estructuras virales, encapsulando el DNA en la parte interna de las estructura y protegiéndolo a su vez del ataque de nucleasas externas. Sin embargo, es necesario añadir un paso de hidrolisis con DNasa y RNasas en la purificación de nanoparticulas que contienen dominios de unión a ácidos nucleicos ya que se ha visto que unen ácidos nucleicos bacterianos provenientes del sistema de expresión bacteriano utilizado para su producción recombinante, afectando muy negativamente sobre su funcionalidad como virus artificiales.
Por lo tanto, dado la gran biocompatibilidad que a priori se espera de las proteínas, sus propiedades arquitectónicas regulables y la posibilidad de funcionalizarlos con ligandos específicos, hace que las nanoparticulas proteicas autoensamblables sean una herramienta muy prometedora para la entrega dirigida de ácidos nucleicos y drogas terapéuticas en las células de cáncer metastatico de colon y en general en las células de mamífero.Cancer is ranked as the second leading cause of death worldwide. Consequently there is a huge necessity of finding more effective cancer therapies. Currently available cancer therapies, far from being effective, present high systemic toxicity and low patient survival rates being the main mortality cause the appearance of metastatic foci, especially in colon cancer. Thus, improving cell specificity and avoiding metastases generation are the mayor challenges for future cancer therapies. In this context, gene therapy appears as very promising alternative therapy since cell targeted personalized therapies can be performed with low systemic toxicity. Since biosafety is the current mayor concern in this type of therapies, multifunctional proteins appear as the most promising gene therapy vectors because of their high biocompatibility and biosafety, low toxicity and really complete tuneability. Moreover, the necessity of effectively controlling nanoparticles size for their efficient biodistribution and delivery has been widely described in the literature. In the present work has been explored the possibility of effectively modulating the self-assembling of multifunctional protein building blocks into predefined size distribution nanoparticles in order to get the full potential of those protein-only nanoparticles for their application in therapeutic drugs and nucleic acids delivery approaches. In this regard, we have described cationic architectonic tag pairs ( one of them being a poly-histidine) that when incorporating to proteins, they induce the self-assembling of protein monomers into nanoparticles with predefined structural properties. It has also been studied the interaction and intracellular trafficking of these kind of protein nanoparticles in cultured cells proving not to be toxic for mammalian cells. Moreover, the structural stability of generated nanoparticles upon intravenous administration in mice has been also studied proving in vitro generated intermolecular interactions during protein assembling process strong enough to ensure nanoparticles structural stability in vivo. It has been shown that CXCR4 chemoquine receptor is a key element in metastasis formation during cancer evolution in different types of tumors, including colorectal cancer, for which metastatic intracellular targeting vehicles are currently missing. In this context, the possibility of effectively functionalizing protein nanoparticles with CXCR4 specific ligands has been deeply explored in this study. Among tested peptide ligands, T22 peptide has shown to be an unusually powerful tag for selective intracellular targeting in CXCR4+ cells having T22-empowered protein nanoparticles of optimal size selectively biodistribute in CXCR4+ cells in vivo. Finally, the suitability of functionalized self-assembling protein nanoparticles for their use as artificial viruses has been also extensively explored. Protein nanoparticles that contain nucleic acid binding domains have shown an appealing capacity to generate virus-like structures when combined with external DNA, completely shielding cargo DNA in the inner part of the structure and protecting it from external nucleases hydrolysis. However, the necessity of an additional DNase/RNase hydrolysis treatment during the nanoparticles purification process has been described since nanoparticles with nucleic acid binding domains have been shown to bind nucleic acids from the bacterial host used for their recombinant expression, resulting strongly detrimental for their functionality as artificial viruses. All together, the high biocompatibility expected for proteins, their regulatable architectonic properties and their efficient targeting possibility, make self-assembling protein-only nanoparticles a very promising material for the therapeutic delivery of drugs and nucleic acids in metastatic colorectal cancer cells and in general in mammalian cells.
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Gopinath, Puja Gopinath. "A Review of Pricing and Reimbursement for Abeona Theraputics’ Gene Therapy Products to Treat Sanfilippo Syndrome." Case Western Reserve University School of Graduate Studies / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=case1497024647261096.
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Oliveira, Franciele de Paula Pansani [UNESP]. "Síntese, caracterização e estudo físico-químico das nanopartículas formadas pela interação de amino derivados de quitosana com DNA." Universidade Estadual Paulista (UNESP), 2011. http://hdl.handle.net/11449/97737.
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oliveira_fpp_me_sjrp.pdf: 646428 bytes, checksum: d3a6b5304dc8d88d292ec3eff61b64ce (MD5)O presente trabalho apresenta a síntese e caracterizaçao de derivados de quitosana obtidos pela reação com cloreto de 2-cloro dietilaminoetilamônio (DEAE Cl). Os derivados obtidos foram utilizados na síntese de nanopartículas para liberação controlada de genes. Inicialmente a quitosana comercial foi desacetilada e as sínteses foram realizadas em meio aquoso alcalino pela reação de substituição nucleofílica dos grupos amino da quitosana. A caracterização dos derivados foi realizada utilizando-se a espectroscopia de ressonância magnética nuclear de prótons (RMN-1 H), titulações potenciométricas e condutimétricas. Os derivados obtidos apresentaram proporções crescentes de DEAE (15%, 24%, 80% e 117%) e diferentes capacidades de tamponamento. A massa molecular viscosimétrica foi determinada por medidas de viscosidade e variou de 17 a 21 kDa. As titulações potenciométricas mostraram que a modificação de quitosana com o grupo DEAE permite aumentar a capacidade tamponante com respeito à quitosana e pode contribuir para aumentar a liberação de DNA e a eficiência do processo de transfecção gênica. O estudo dos derivados com DNA foi realizado utilizando-se as técnicas de fluorescência, eletroforese, espalhamento de luz dinâmico (tamanho das nanopartículas) e potencial zeta. O ensaio com brometo de etídio mostrou que a força de interação de DNA com os derivados depende tanto do grau de substituição por DEAE quanto da razão de cargas (N/P). Em altos valores de pH (7,4) todos os derivados interagem fortemente com DNA devido a presença dos grupos amino terciários e quaternários ligados a cadeia de quitosana. Os resultados de eletroforese mostram que a força de interação aumenta com o grau de substituição de DEAE e os resultados iniciais de espalhamento de luz mostram que nanopartículas estáveis podem ser obtidas em pH fisiólogico...
This work presents the synthesis and characterization of chitosan derivatives obtained by reaction of chitosan with diethylaminoethyl chloride - Cl DEAE. The obtained derivatives were employed in the synthesis of nanoparticles for controlled delivery of genes. Commercial chitosan was deacetylated and the reactions were carried out in aqueous solution at pH 8.0 by the nucleophilic subsitituion of the chitosan amino groups. The characterization was performed using 1H NMR spectroscopy, potentiometric and conductimetric titrations. The obtained derivatives contain increasing proportions of DEAE (15%, 24%, 80% and 117%) and different buffering capacities. Viscosity measurements were performed to determine the viscoimetric molecular weight (Mv), which varied from 17 to 21 kDa. The potentiometric titrations showed that DEAE groups increase the buffering capacity of chitosan and this may contribute to increase the DNA delivery from nanoparticles and the efficiency of gene transfection. The study of interaction between the derivatives and DNA was performed using fluorescence, gel electrophoresis, dynamic light scattering and zeta potential. The ethidium bromide (EtBr) assay showed that the strenght of interaction between the derivatives and DNA depends on both, the degree of substitution (DS) and N/P charge ratio. At physiological pH all derivatives interacted with DNA due to the presence of tertiary quaternary amino groups attached to the chitosan main chain. The gel electrophoresis results showed that the strength of interaction increases with DS and the initial results employing light scattering techniques show that stable nanoparticles can be obtained in phyisological pH, which is clearly an important property in comparison to deacetylated chitosan. Transfection studies carried out with these derivatives confirmed that the presence of DEAE groups improves the transfection efficiency... (Complete abstract click electronic access below)
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BASÍLIO, João Paulo Santana. "Genetic engineering of human cell lines for the improvement of viral vector production for gene therapy." Master's thesis, Instituto de Higiene e Medicina Tropical, 2018. http://hdl.handle.net/10362/61545.
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A terapia génica baseada em vetores virais toma partido dos mecanismos biológicos naturais para entregar genes terapêuticos e controlar a sua expressão nas células alvo do paciente. Vários produtos de terapia génica viral estão já no mercado, com cerca de metade destes utilizando vírus recombinantes da família Retroviridae. Estes são uma opção frequente devido à sua elevada capacidade de transporte de carga genética, elevada eficiência de transdução, integração estável de genoma em loci transcricionalmente ativos, quer as células estejam em divisão ou não e devido à expressão a longo termo do gene entregue. As previsões de receita no mercado de terapia génica viral até 2020 ultrapassam os 200 milhões de dólares, sem perspetivas de declínio pelo menos até 2026. No entanto, a produção de retrovírus recombinantes depara-se com vários desafios. A elevada produção de partículas não-infecciosas – aproximadamente 1 em cada 1000 partículas produzidas são infecciosas – e baixo rendimento das plataformas de produção, dois fatores que impõem custos de produção altos, são as barreiras mais difíceis de ultrapassar na transição clinico-mercado.
Vias metabólicas recrutadas em produção de retrovírus recombinantes foram previamente identificadas. Neste trabalho, cinco genes-alvo foram alvo de estudo numa linha celular produtora de retrovírus recombinantes. A sua sobre-expressão foi realizada através de transdução com vetores lentivirais. Os cinco genes considerados – BCL2, GSR, HSPA5, PDIA e XBP1 – pertencem a vias envolvidas em anti-apoptose, metabolismo de glutationa e síntese proteica no retículo endoplasmático. As populações resultantes foram caracterizadas quanto a crescimento celular, produtividade viral, expressão dos componentes virais e dos genes metabólicos.
Verificou-se um aumento de produtividade associada à sobre-expressão dos genes intervenientes na síntese proteica no retículo endoplasmático. Aumentos de 140% foram obtidos com o gene XBP1 – que está associado à resposta de proteínas mal conformacionadas. Aumentos mais modestos, na ordem dos 63% foram obtidos com o gene PDIA2 – que está associado à catálise de pontes dissulfureto. Por último, aumentos de 76% foram observados com HSPA5 – que está associado a um grande leque de processos envolvidos em síntese proteica no retículo endoplasmático. Não foram observados aumentos de produção com genes associados à anti-apoptose nem ao metabolismo de glutationa, nomeadamente BCL2 e GSR.
Os resultados aqui obtidos demonstram que engenharia metabólica é uma estratégia valiosa para o melhoramento de produção de retrovírus recombinantes. Três dos cinco genes abordados levaram ao aumento de produção de retrovírus recombinantes, o que apoia a síntese proteica como um alvo valioso na resolução dos constrangimentos encontrados na produção de retrovírus recombinantes. Este trabalho contribui para o campo da terapia génica baseada em vetores virais. O conhecimento gerado neste trabalho é relevante para outros vetores virais e para engenharia metabólica de linhas celulares humanas.Gene therapy using viral vectors harnesses naturally occurring viral biological mechanisms to deliver therapeutic genes and control their expression in patient target cells. Several viral gene therapy products have already reached the market, with nearly half being based on recombinant viruses belonging to the Retroviridae family. These are a frequent option since they have large genetic payload capacity, high transduction efficiency, stable genome integration in transcriptionally active loci of dividing and non-dividing cells and sustained long-term expression of the delivered gene. Revenue predictions for viral gene therapy market in 2020 surpass 200 million US dollars, with no decline perspective until at least 2026. Yet, recombinant retroviral synthesis faces several challenges. High non-infective particle concentration – roughly 1 in every 1000 produced particles are infective – and low yields of current production platforms, both of which impose high production costs, present the hardest barriers to overcome in clinical to market transition. Previously, metabolic pathways recruited in recombinant retroviral production were identified. In this work, five target genes were overexpressed in a stable recombinant retrovirus producer cell line through lentiviral vector transduction with incremental target gene expression. The five considered genes – BCL2, GSR, HSPA5, PDIA and XBP1 – belong to pathways involved in anti-apoptosis, glutathione metabolism and endoplasmic reticulum protein synthesis. Resulting populations were characterized for cell growth, recombinant retrovirus productivity, viral components and metabolic gene expression. Increase in productivity was associated to overexpression of genes intervenient in endoplasmic reticulum protein synthesis. Increases of 140% were obtained with XBP1 gene – which is associated to unfolded protein response and thus, correct protein folding. More modest increases of 63% were attributed to PDIA2 gene – which is associated to disulfide bond catalysis. Lastly, increases of 73% can be observed with HSPA5 – which is associated to a wide range of protein synthesis processes within the endoplasmic reticulum. Improvements in productivity were not observed with anti-apoptotic nor glutathione associated genes, namely BCL2 and GSR. The results herein obtained demonstrate cell metabolic engineering as a valuable strategy to improve recombinant retroviral production. Three of the five targeted genes resulted in higher recombinant retroviral production supporting protein synthesis as powerful targets for debottlenecking recombinant retroviral production. This work contributes for the viral gene therapy field. The knowledge generated in this work is relevant to other viral vectors and for metabolic engineering of human derived cell lines.
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Morin, Nicolas. "Expression of mutated HIV-1 Gag-Pol proteins and their effects on virus replication and infectiousness, implications for gene therapy." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0015/MQ37152.pdf.
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Villalobos, Alberú Xenia. "Polypurine Reverse Hoogsteen hairpins: stability, lack of immunogenicity and gene silencing in cancer therapy." Doctoral thesis, Universitat de Barcelona, 2015. http://hdl.handle.net/10803/360588.
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This work is focused on the study of the stability and immunogenic properties of the Polypurine Reverse Hoogsteen hairpins (PPRHs), and on their use as a gene-silencing tool. PPRHs are non-modified DNA molecules formed by two antiparallel polypurine strands linked by a pentathymidine loop that allows the formation of intramolecular reverse Hoogsteen bonds between both strands. Previously in our laboratory it was demonstrated that these hairpins bind to their polypyrimidine target in a dsDNA via Watson-Crick bonds, displacing the polypurine strand of the target duplex. The effect of PPRHs in cells and their mechanism of action were first described using PPRHs designed against the template and coding strands of the DHFR gene. A PPRH against survivin was further validated in a xenograft tumor model, establishing the proof of principle for the use of PPRHs as a therapeutic tool.
In this work we increased the knowledge we have about PPRHs. We were able to establish that PPRHs, unlike siRNAs, are very stable molecules in different types of serum and inside the cells. We also established that PPRHs do not induce the innate immune response, since they do not induce the levels of neither the transcription factors IRF3 and NF-κB, nor the proinflammatory cytokines IL-6, TNF-α, IFN-α, IFN-β, IL-1β, and IL-18. Additionally, unlike siRNAs, PPRHs did not trigger the activation of the inflammasome. Another element that we studied was the modification of the PPRH structure, since it has been shown that circular structures can provide advantages over linear structures. Therefore, we analyzed the efficacy of two other types of PPRH: i) nicked-circle-PPRHs, a new structure in which a second loop was introduced to form a nearly circular sequence, and ii) PPRHs made out of RNA (RNA-PPRHs).
To broaden the applicability of PPRHs in cancer therapy, we evaluated their capacity to silence genes involved in a variety of biological functions linked to cancer hallmarks. The genes selected were: BCL2, MDM2, MYC, TOP1 and MTOR, and the validation of the PPRHs was performed in different cancer cells lines (PC3, MIA PaCa2, HCT116, SKBR3, MCF7 and MDA-MB-468). Regardless of the gene or cell line tested, PPRHs were able to decrease cell survival and mRNA expression levels, and to increase apoptosis, to a greater or lesser extent. Finally, we also present an approach to increase the specificity of PPRHs that involves the use of a DNA aptamer that has been shown to have an effect in HER2 positve cells.Este trabajo se centra en el estudio de la estabilidad y potencial inmunogénico de las moléculas llamadas PPRHs (Polypurine Reverse Hoogsteen hairpins), y en su uso como herramienta de silenciamiento génico. Los PPRHs son moléculas de DNA no modificadas. Están formadas por dos cadenas antiparalelas de polipurinas unidas por un bucle pentatimidínico, lo que permite la formación intramolecular de enlaces de Hoogsteen reversos entre ambas cadenas. Anteriormente se demostró que los PPRH son capaces de unirse a una secuencia de dsDNA mediante enlaces de Watson-Crick, formando un triplex que desplaza la cadena polipurínica de la diana. La prueba de principio para el uso de los PPRH como agente terapéuticos se llevó a cabo con un PPRH dirigido al gen de la survivina el cual fue validado tanto in vitro como in vivo. En esta tesis se ha profundizado en el conocimiento de los PPRHs. Se ha establecido que estas moléculas son estables en diferentes tipos de suero, así como intracelularmente. Además se determinó que los PPRHs, no inducen la activación de la respuesta inmune innata, ya que su transfección a una línea monocítica no incrementó los niveles de expresión de los factores de transcripción IRF3 y NF-κB, y tampoco de las citoquinas proinflamatorias IL-6, TNF-α, IFN-α, IFN-β, IL-1β, y IL-18. Además, los PPRHs no indujeron la activación del inflamasoma. También se diseñaron dos estructuras nuevas de PPRH: PPRH circulares y PPRHs basados en RNA, y se estudiaron diversos parámetros como su capacidad de unión a la diana y su citotoxicidad. Para ampliar la aplicabilidad de estas moléculas, se validó el uso de los PPRHs sobre diversos genes relevantes en cáncer: BCL2, MDM2, MYC, TOP1 y MTOR. Esta validación se realizó en diversas líneas tumorales (PC3, MIA PaCa-2, HCT116, SKBR3, MCF7 y MDA-MB-468) observando que los PPRHs fueron capaces de disminuir la supervivencia de las células y la expresión de los genes diana, y de aumentar la apoptosis. Finalmente, se realizó una aproximación para incrementar la especificidad de los PPRHs, la cual incluye el uso de un aptámero de DNA dirigido a la proteína de membrana HER2.
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Lang, Annemarie [Verfasser]. "Reviewing Challenges in Osteoarthritis Gene Therapy and Introduction of a Molecular Therapeutic Approach in an Equine Model System / Annemarie Lang." Berlin : Freie Universität Berlin, 2016. http://d-nb.info/1099282764/34.
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Knoop, Kerstin. "Molecular imaging and radionuclide therapy in non-thyroidal tumors after mesenchymal stem cell- mediated sodium/iodide symporter (NIS) gene transfer." Diss., Ludwig-Maximilians-Universität München, 2014. http://nbn-resolving.de/urn:nbn:de:bvb:19-178550.
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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.
Full textAbstract:
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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Alamoudi, Aliaa. "Regulated antagonism of immune suppressive molecules in tumours." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:95599708-58e8-4a35-92b9-4e31523556f3.
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Despite expressing antigens that can induce immune surveillance and immune eradication, tumours demonstrate the capacity to evade anti-tumour immunity. Recently, this has been attributed to the ability of tumours to induce a local immunosuppressed micro-environment, which is a major obstacle to successful natural and vaccine induced anti-tumour immunity. Soluble factors such as transforming growth factor beta (TGFβ), and interleukin 10 (IL-10), released by cancer and stromal cells, are thought to play a significant role in this local immunosuppression. In order to assess the influence of antagonising these soluble factors locally on tumour biology and tumour immunity, a murine CT26 colorectal carcinoma model that can express cytokine antagonists under Doxycycline (Dox) control was engineered. Two stable CT26 cell lines expressing Dox-inducible soluble extracellular domain of TGFβ receptor II (STGFβRII) or soluble extracellular domain of IL-10 receptor (SIL-10R), were established. Expression of STGFβRII in vitro and in vivo was only evident after Dox treatment. When Dox was administered directly following subcutaneous (s.c.) inoculation of STGFβRII-expressing CT26 cells into Balb/c mice, tumour growth was significantly suppressed. Interestingly, inducing STGFβRII in well-established tumours showed less suppression of tumour growth. To assess the effect of expressing STGFβRII on tumour immunity the RNA expression of 22 common cytokines was measured in the tumours of mice receiving Dox and control mice. The levels of some of these cytokines were modulated by STGFβRII expression (e.g TGFβ,Tnfsf9, IL-2). We also tested for any additive effect between expressing STGFβRII, and the administration of anti-CTLA-4 antibody on tumour growth, and tumour immunity. The model described here could help address various limitations seen previously in studies of TGFβ blockade. It provides means of effective local antagonism, and it addresses immunological endpoints which have been limited in previous studies. Because of its tight regulation, the model also allows identification of the best timing of TGFβ blockade alone or in combination with other immunotherapeutics.
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Singh, Preetinder Pal Clinical School Prince of Wales Hospital Faculty of Medicine UNSW. "A combination of molecular and traditional chemotherapy: prospects of synergies against cancer." Awarded by:University of New South Wales. Clinical School - Prince of Wales Hospital, 2009. http://handle.unsw.edu.au/1959.4/44564.
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In this study, we have explored the combination of a novel Purine Nucleoside Phosphorylase mediated Gene-Directed Enzyme Prodrug Therapy (PNP-GDEPT) with chemotherapeutics, Taxotere and/or Carboplatin to target prostate and ovarian cancer (PC & OC). PNP converts the prodrug (Fludarabine-phosphate) to a toxic purine, 2-fluoroadenine (2FA) that inhibits RNA/DNA synthesis. Taxotere is active against late stage PC whilst carboplatin is first line therapy for OC. Neither modality is adequately effective. We expect that a combination will target heterogeneity via cytotoxicity to diverse cancer cell populations leading to effective synergies, which may improve efficacy and quality of life. For PC, Synergy between Ad-PNP-GDEPT and Taxotere were assessed in vitro and in vivo. Cell killing effects of combination led to significant synergistic killing of human PC-3 & murine RM1 PC cells accompanied by enhanced apoptosis. A lower individual dose (by up to 8 fold) led to enhanced efficacy. In vivo, the combination regimen given at the suboptimal doses led to reduction in local tumour (PC-3 & RM1) growth in nude and in C57BL/6 mice, respectively. A significant reduction in lung RM1 colony numbers indicated enhanced systemic efficacy. Combination treated mice also displayed significantly improved survival (25 days vs 15 days for control mice). Importantly, the condition of combination treated mice (e.g. weight loss) was better than those given individual treatments. The possible involvement of the immune system in this enhanced effect is under investigation. For OC, three-way synergy between Ad-PNP-GDEPT, Taxotere and carboplatin was effectively demonstrated in SKOV-3 and OVCAR-3 cells. This was significantly greater than bimodal or individual treatments. A 10-50 fold dose reduction of individual treatments was effective when combined, accompanied by enhanced apoptosis. Western-blotting analyses revealed a shift in the expression of anti-apoptotic and proapoptotic proteins upon treatment with various combinations. This is the first demonstration of synergy between these modalities.
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Salazar, Marcela d'Alincourt. "Genomic Effects of Hormonal Adjuvant Therapies that Could Support the Emergence of Drug Resistance in Breast Cancer." University of Toledo Health Science Campus / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=mco1280929084.
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Marrero, Bernadette. "Evaluation of Immunogene Therapy Using a Plasmid Encoding IL-15 Delivered by Electroporation in a 3D Tumor Model and a Mouse Melanoma Model." Scholar Commons, 2010. http://scholarcommons.usf.edu/etd/3520.
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Melanoma is an aggressive disease with few effective treatment options. Non-toxic, anti-tumor therapies and prophylactic approaches are currently being investigated to identify treatment options that will control and remove late-stage melanoma.
The overall goal of this project was to establish an effective delivery method for a plasmid encoding human interleukin (phIL-15) into mouse melanoma cells (B16.F10) using the gene transfer technique electroporation (EP)1. The EP delivery phIL-15 was optimized using an in vitro 3D tumor model. The purpose was to translate these IL-15 delivery conditions into an in vivo mouse melanoma model to study IL-15 signal transduction and stimulate immune cells to destroy tumor antigens as well as promote an anti-tumor immune memory response.
The in vitro 3D tumor model and the mouse model demonstrated similar expression patterns when delivering phIL-15 with different EP conditions. Intra-tumoral delivery using 500V/cm 20ms enhanced gene delivery and increased IL-15 protein expression compared to 1300V/cm 100μs. There was also a visible increase in transfection efficacy between tumor cells compared to skin cells when delivering pmIL-12 and phIL-15 plasmid constructs in vivo. The plasmid+EP groups 1300V/cm and 500V/cm stimulated increased expression of cytokines IL-1β, IL-6, INFγ, MIP-1β and TNFα. These EP groups also promoted tumor regression by up-regulating CD8+ T cells and CD4+ T cells which targeted melanoma. Regression and survival studies demonstrated that 73.3% of mice cleared B16.F10 cells when treated with phIL-xi15+1300V/cm and pVax+500V/cm. In addition, 53% of the mice responded to the phIL-15+500V/cm treatment group. Furthermore, 75% of the mice from group phIL-15+500V/cm survived secondary inoculation and tumor challenge. In conclusion, plasmid with encoding gene insert phIL-15 delivered by EP has the potential to act as an anti-tumor therapy because it promotes melanoma regression and enhances mouse survival through innate and adaptive cell-mediated immune responses.