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Dissertationen zum Thema „Gene therapy“
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1
Vasanwala, Farha Huseini. „Gene manipulations for cancer gene therapy“. Diss., The University of Arizona, 2002. http://hdl.handle.net/10150/289776.
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Tumor cells can be modified with cytokine genes such as the Interleukin-2 (IL-2) gene. The levels of IL-2 expressed are critical for successful treatment. We have tried to achieve higher levels of IL-2 than those currently available by conventional plasmids. Use of a transcriptional activator, e.g; the tat gene along with the HIV promoter driving the IL-2 gene, greatly increased IL-2 levels compared to widely used cytomegalovirus (CMV) driven plasmids. Control of the tat gene with an inducible promoter, i.e; the human HSP70B promoter, permitted control of gene expression. The inducibility of the HSP70B promoter by heat, γ-radiation and geldanamycin (a chemotherapeutic drug) allowed for a combinatorial approach to cancer treatment with hyperthermia, radiation therapy and chemotherapy. Also a brief heat treatment of 10 min at 42°C of target cells increased plasmid uptake, and higher levels of gene expression could be achieved. Another arm of immunotherapy is adoptive therapy with Tumor Infiltrating Lymphocytes (TILs). Insufficient numbers of tumor-specific T-cells limit the success of TIL therapy. An alternative approach to overcome this limitation is to transfer tumor-specific T cell receptor (TCR) into peripheral T-cells, redirecting their specificity to the tumor cell. To prove the feasibility of this technique, T-cell receptors were identified and cloned from hybridomas specific for the tumor cell line, MO5. A three domain single chain T-cell receptor was also constructed from the tumor-specific TCR genes to investigate the ability of a single chain T-cell receptor to activate T-cells. The CD3ζ chain was linked to the single chain to allow signal transduction upon antigen recognition by the TCR. The full length and the single chain TCR were cloned into a retroviral vector and transfected into mouse and human T cell lines. Cell surface expression of the chains were detected by flow cytometry. Functionality of the transfected TCR chains was assessed by IL-2 secretion on co-culture of the tumor cell line MO5 and the transfected T-cells. The two different approaches described here, i.e; higher levels of IL-2 for IL-2 gene therapy and specific redirection of T-cells can potentially greatly enhance the success rate of cancer treatment.
2
Santos, João Miguel Almeida. „Gene therapy: development of a new nanocarrier system for mitochondrial gene therapy“. Master's thesis, Universidade da Beira Interior, 2013. http://hdl.handle.net/10400.6/1627.
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Mitochondria are unique organelles that have their own genome, the mitochondrial
DNA (mtDNA). Although quite small compared to nuclear DNA (nDNA), mutations in mtDNA are
quite frequent due to the lack of protection and repair mechanisms. Per consequence,
cytopathies and diseases are quite common and mostly associated with high energy
demanding tissues such as muscles and the brain. Therefore, the development of a new and
efficient mitochondrial gene therapy protocol is seen as a promising approach.
During this MSc thesis we try to bring together a new nanocarrier system with the
ability to deliver plasmid DNA into the mitochondria, for future application in mitochondrial
gene therapy (MGT).
Hence, the development of this research project can be divided itself into three main
stages:
1. Isolation and purification of three plasmid DNAs (pUC19, pVAX1-LacZ and
pcDNA3-myc-FLNa S2152A);
2. Synthesis and characterization of nanoparticles with mitochondria affinity;
3. In vitro study of mitochondrial transfection ability.
The newly developed nanoparticles, created through a co precipitation method, offer
us unique features such as: biocompatibility, plasmid DNA (pDNA) encapsulation efficiency
and low manufacturing cost.
We were able to successfully achieve transfection into the mitochondria which may
result in a huge step in the correction of mitochondrial defects, offering new therapeutic
strategies for a variety of pathologies ranging from cancer to Parkinson and Alzheimer´s
diseases.As mitocôndrias são organelos únicos pois possuem o seu próprio genoma, o ADN mitocondrial (ADNmt). Apesar de bastante pequeno quando comparado com o ADN nuclear (ADNn), mutações ao nível do ADNmt são bastante frequentes devido à falta de mecanismos de protecção e de reparação. Como consequência, citopatias e doenças associadas à mitocôndria são bastante frequentes afectando essencialmente órgãos e tecidos onde existe muito dispêndio de energia como é o caso dos músculos e do cérbero. Logo, o desenvolvimento de um novo e eficiente protocolo para terapia génica mitocondrial (MGT) é visto como uma proposta aliciante. Durante esta tese de Mestrado, tentamos criar um novo nanosistema que consiga entregar eficazmente ADN plasmídico (pDNA) à mitocôndria para que no futuro possa ser usado em terapia génica mitocondrial (MGT). Assim, este projecto de investigação pode ser dividido em três etapas principais: 1. O isolamento e purificação de três plasmídeos (pUC19, pVAX1-LacZ e pcDNA3-myc-FLNa S2152A); 2. A síntese e caracterização de nanopartículas com afinidade para a mitocôndria; 3. O estudo da capacidade das nanopartículas efectuarem transfecção celular e dirigirem-se à mitocôndria; As nanopartículas desenvolvidas, através do método de co-precipitação oferecem-nos qualidades únicas como a sua biocompatibilidade, alta eficiência de encapsulamento de ADN e baixo custo de produção. A transfecção celular foi alcançada com sucesso sendo que, tais resultados, podem contribuir em grandes avanços na correcção de defeitos mitocondriais, oferecendo-nos uma nova estratégia terapêutica no combate a diversas patologias desde o cancro, às doenças de Parkinson e Alzheimer.
3
Nanda, Dharminderkoemar. „Gene therapy for gliomas“. [S.l.] : Rotterdam : [The Author] ; Erasmus University [Host], 2008. http://hdl.handle.net/1765/13140.
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4
Hayes, E. A. L. „Anti-angiogenic gene therapy“. Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.603877.
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The aim of this project was to assess a novel anti-angiogenic gene therapy in which a therapeutic gene is activated exclusively in proliferating endothelial cells using tissue-specific promoters and the Cre/IoxP recombination system. Adenoviruses and transgenic mice were generated in parallel to test individual components of the targeting system. One of the therapeutic effector strategies investigated was the herpes simplex virus-1 thymidine kinase (HSV-1TK)/ganciclovir (GCV)-mediated suicide system, which is reported to kill proliferating cells selectively. Administration of GCV to pTie2-TK transgenic mice expressing HSV-1 TK under the control of the endothelial cell-specific Tie2 promoter and to mice treated systemically with a pTie2-TK adenovirus was lethal, demonstrating that additional control was required to target exclusively proliferating endothelial cells. Intra-tumoural (i.t.) injection of pTie2-TK adenovirus resulted in tumour-restricted expression of HSV-1 TK and preliminary data demonstrated a trend towards a decrease in primary tumour growth following treatment of mice with i.t. pTie2 adenovirus and GCV. The tet Off system was investigated as a method to obtain conditional control of Cre recombinase expression. Despite showing tight regulation in vitro, this system did not result in complete silencing of transgene expression in vivo. Transgenic mice expressing tamoxifen (TMX)-regulated Cre recombinase under the control of the cell-cycle dependent Cyclin A promoter were also generated, but problems with TMX administration precluded determination of whether Cre recombinase was activated by TMX in these mice. However, conditional transgene activation was achieved in vivo by generating a pCycA-Cre adenovirus in which the Cyclin A promoter was used to drive expression of Cre recombinase. A pTie2-stuffer-TK transgenic mouse line was generated which expressed a Cre-activatable form of HSV-1 TK under the control of the Tie2 promoter. To target proliferating endothelial cells specifically, the pCycA-Cre adenovirus was used to activate HSV-1 TK in these transgenic mice. Preliminary data showed that there was a trend towards a decrease in primary tumour growth following treatment of pTie2-stuffer-TK transgenic mice with i.t. pCycA-Cre adenovirus and GCV. Transgenic mice expressing an alternative Cre-activated therapeutic gene, the pro-apoptotic gene Bax, under the control of the Tie2 promoter were generated by direct pronuclear injection and by site-specific transgene insertion into the hprt locus. pTie2-stuffer-Bax mice generated using the latter technique showed higher levels of Tie2 promoter-driven transgene expression than those made by pronuclear injection.
5
Bilsland, Alan. „Telomerase directed gene therapy“. Thesis, University of Glasgow, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.272871.
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6
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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7
Morin, Kevin Wayne. „Scintigraphic imaging during gene therapy“. Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq21605.pdf.
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8
Singwi, Sanjeev. „HIV gene therapy using nucleases“. Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0001/MQ46100.pdf.
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9
Horst, Maarten ter. „Gene therapy of malignant gliomas“. [S.l.] : Rotterdam : [The Author] ; Erasmus University [Host], 2008. http://hdl.handle.net/1765/10864.
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10
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
11
Harris, Jonathan David. „Targeted gene therapy for cancer“. Thesis, Imperial College London, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.309239.
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12
Rigg, Anne Sagar. „Gene therapy for human cancer“. Thesis, Imperial College London, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.341902.
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13
Pandha, Hardev Singh. „Gene transfer therapy for cancer“. Thesis, Imperial College London, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299872.
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14
Page, Sean Michael. „Gene therapy for haemophilia B“. Thesis, University of Oxford, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.318970.
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15
White, Stephen John. „Ex vivo keratinocyte gene therapy“. Thesis, University of Oxford, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.268103.
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16
Bainbridge, James William Braithwaite. „Gene therapy for ocular angiogenesis“. Thesis, University College London (University of London), 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.409631.
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17
Miller, Gaynor. „Modelling gene therapy for haemophilia“. Thesis, University College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.369099.
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18
Roeder, Geraldine Elizabeth. „Gene therapy for cervical cancer“. Thesis, University of Bristol, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.268704.
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19
Knight, S. B. „Lentiviral vectors for gene therapy“. Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1346459/.
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Lentiviral vectors, derived from HIV-1, are promising tools for gene therapy. Recent clinical trials have demonstrated the translation of their effectiveness in laboratory studies to clinical trials. However there are still limitations, relating to vector safety and efficiency of production, that could confine their future use. I investigated the ability of lentiviral vectors to perturb cellular gene expression by insertional mutagenesis (IM), using an in vitro model that detects aberrant splicing from lentiviral vectors to the growth hormone receptor gene (Ghr). The lentiviral vector pHV with full long terminal repeats (LTRs) and an internal spleen focus forming virus promoter (SFFV), was previously found to activate Ghr expression by a fusion mRNA transcript initiated in the HIV LTR (46). I extended this discovery to show that the SFFV promoter was enhancing expression from the HIV LTR, leading to IM (269). Application of our in vitro IM assay to potential clinical lentiviral vectors revealed that the novel ‘UCOE’ (ubiquitously acting chromatin opening element) promoter, within a selfinactivating (SIN) lentiviral vector, could drive UCOE-Ghr mRNA transcripts, causing IM. Mutation of splice donor sites in UCOE alone was insufficient in abrogating IM, however internal deletions around these splice donor sites were more successful. In other work, I made a packaging cell line for lentiviral vectors by stably expressing rev and a modified RD114 env (RDpro) in a cell line expressing HIV gag-pol. This led to the isolation of 57R10E, a cell line that made a titer of over 104 infectious units per ml when a SIN lentiviral vector was transiently or stably expressed. Taken together, this work will broaden the application of lentiviral vectors in clinical gene therapy by reducing both the chances of adverse events and the costs associated with vector production.
20
Savina, Yulia. „Gene therapy of Parkinson's disease“. Thesis, Київський національний університет технологій та дизайну, 2019. https://er.knutd.edu.ua/handle/123456789/13159.
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21
Haidet, Amanda M. „Gene Therapy for Neuromuscular Disorders“. The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1270479273.
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22
CATTANEO, STEFANO. „Combinatorial gene therapy for epilepsy“. Doctoral thesis, Università Vita-Salute San Raffaele, 2022. http://hdl.handle.net/20.500.11768/128275.
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Epilepsy is a neurological disease characterized by a persistent predisposition to generate seizures, that affects about 1% of the world population. About 30% of epileptic patients are drug-resistant, thus refractory to currently available anti-epileptic drugs (AEDs). Less than 10% of these drug-resistant patients are eligible for resective brain surgery, often due to generalized or multiple epileptic foci, or due to proximity of the epileptic focus to eloquent brain areas. Therefore, gene therapy may represent a doable approach for the unmet medical need of these patients.
Neuropeptide Y (NPY) can act as an endogenous anticonvulsant. NPY expression is increased both in rodent and human hippocampal sections from temporal lobe epilepsy surgical samples, despite the strong loss of hilar GABAergic interneurons. Therefore, NPY-based gene therapy may represent a novel approach for the treatment of focal epilepsies. Ideally, however, such vectors should contain multiple elements (at least NPY and Y2Rs driven by appropriate promoters).
In the past, great advancements in the field of viral vectors based on HSV-1 have been made by our laboratory. We therefore aimed at combining the potential of HSV vectors to accommodate large payloads with the complexity of the NPY system to create an “ideal” combinatorial therapeutic cassette. However, residual concerns on the safety and translatability of our new generation HSV-1 based vectors (named J∆NI8) let us first characterize their electrophysiological properties in primary neuronal culture, to assess both safety and efficacy profiles. Surprisingly and disappointingly, we show that mutations in the envelope glycoprotein B (gB), which is responsible for viral entry and cell fusion, might arise during viral vector production. In turn, mutated gB can increase firing frequency while reducing both input resistance and resting membrane potential of transduced neurons. Altogether, these data suggest that careful evaluation of envelope glycoproteins is needed to develop safe HSV-1 replication-defective vectors for the treatment of CNS disorders. We, therefore, decided to move to LV vectors, a more robustly characterized platform despite a more limited packaging capacity compared with HSV vectors.
To potentiate the protective effect of NPY, we developed a combinatorial gene therapy approach based on the expression of NPY together with its receptor (Y2). Since Y2 receptors act mainly pre-synaptically to reduce glutamate release by lowering Ca2+
influx, transgenes expression was driven by the minimal CamKII promoter, thereby biasing their expression in excitatory neurons. We characterized the ability of our lentiviral vectors to express NPY and its functional Y2 receptor in hippocampal neurons and mouse brains. Telemetry video-EEG monitoring was then used to assess the effect of the therapeutic genes on the epileptic phenotype of a genetic mouse model of epilepsy.
We found that the combined expression of NPY and Y2 is sufficient to reduce both the frequency and duration of seizures in the Synapsin triple-KO epilepsy model. These data further strengthen the hypothesis that strategies aimed at the delivery of NPY and Y2 may be successful for the treatment of epilepsy, particularly for pharmaco-resistant and genetic forms of the disease.L'epilessia è una malattia neurologica caratterizzata da una persistente predisposizione a generare crisi, che colpisce circa l'1% della popolazione mondiale. Circa il 30% dei pazienti epilettici sono resistenti ai farmaci, quindi refrattari ai farmaci antiepilettici attualmente disponibili (AED). Meno del 10% di questi pazienti resistenti ai farmaci sono eleggibili per la chirurgia, spesso a causa di foci epilettici generalizzati o multipli, o a causa della vicinanza del focus epilettico alle aree cerebrali eloquenti. Pertanto, la terapia genica può rappresentare un approccio fattibile. Il neuropeptide Y (NPY) può agire come un anticonvulsivo endogeno. L'espressione di NPY è aumentata sia nelle sezioni ippocampali di roditori che in quelle di campioni chirurgici umani di epilessia del lobo temporale, nonostante la forte perdita di interneuroni GABAergici a livello dell’ilo. Pertanto, la terapia genica basata su NPY può rappresentare un nuovo approccio per il trattamento delle epilessie focali. Idealmente, tuttavia, tali vettori dovrebbero contenere più elementi (almeno NPY e Y2R guidati da promotori appropriati). In passato, il nostro laboratorio ha fatto grandi progressi nel campo dei vettori virali basati su HSV-1. Abbiamo quindi mirato a combinare il potenziale dei vettori HSV di ospitare DNA di grandi dimensioni, e la complessità del sistema NPY, per creare una cassetta terapeutica combinatoria "ideale". Tuttavia, le preoccupazioni residue in merito alla sicurezza della nostra nuova generazione di vettori basati su HSV-1 (chiamati J∆NI8) ci hanno spinto a valutare i profili di sicurezza ed efficacia in vitro per valutare l’effetto dell’infezione sulle proprietà elettrofisiologiche in neuroni primari. Sorprendentemente e in maniera deludente, abbiamo dimostrato che mutazioni nella glicoproteina B dell'involucro (gB), che è responsabile dell'entrata virale e della fusione cellulare, potrebbero sorgere durante la produzione del vettore virale. A livello elettrofisiologico, abbiamo inoltre visto che la gB mutata può aumentare la frequenza di potenziali d’azione e contemporaneamente ridurre sia la resistenza di ingresso che il potenziale di riposo neuroni trasdotti. Complessivamente, questi dati suggeriscono che un'attenta valutazione delle glicoproteine dell'involucro è necessaria per sviluppare vettori sicuri non replicativi basati su HSV-1 per il trattamento dei disturbi del SNC. Abbiamo quindi deciso di passare ai vettori Lentivirali (LV), una piattaforma più robusta e caratterizzata nonostante una capacità di carico più limitata rispetto ai vettori HSV. Per potenziare l'effetto protettivo di NPY, abbiamo sviluppato un approccio combinatorio di terapia genica basato sull'espressione di NPY insieme al suo recettore (Y2). Poiché i recettori Y2 agiscono principalmente a livello pre-sinaptico per diminuire il rilascio di glutammato riducendo l’ingresso di Ca2+, l'espressione dei transgeni è stata guidata dal promotore minimal CamKII, orientando così la loro espressione selettivamente nei neuroni eccitatori. Abbiamo successivamente caratterizzato la capacità dei nostri vettori LV di esprimere NPY e il suo recettore funzionale Y2 nei neuroni ippocampali e nel cervello dei topi. In seguito, abbiamo utilizzato un sistema di monitoraggio video-EEG mediante telemetria per valutare l'effetto dei geni terapeutici sul fenotipo epilettico in un modello genetico di epilessia. Abbiamo scoperto che l'espressione combinata di NPY e Y2 è sufficiente a ridurre sia la frequenza che la durata delle crisi nel modello di epilessia Synapsin triple-KO. Questi dati rafforzano ulteriormente l'ipotesi che le strategie mirate all’utilizzo di NPY e Y2 possono avere successo per il trattamento dell'epilessia, in particolare per le forme resistenti ai farmaci ma anche per forme genetiche della malattia.
23
Rück, Andreas. „Myocardial gene therapy and gene expression in angina pectoris /“. Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-648-4/.
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24
Abunasra, Haitham Juma. „Gene therapy in myocardial ischemia-reperfusion“. Thesis, Imperial College London, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.404964.
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25
Piccirillo, Ciriaco A. „Cytokine gene therapy of autoimmune disease“. Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape11/PQDD_0023/NQ50237.pdf.
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26
Aints, Alar. „Vector development for suicide gene therapy /“. Stockholm, 2002. http://diss.kib.ki.se/2002/91-7349-199-3.
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27
Samani, Amir Abbas. „IGF-IR targeted cancer gene therapy“. Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=18206.
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Since the declaration of “War on cancer” in 1971, and with the insight provided by recent advances in human genetics and in molecular technology , the field of cancer biology has been expanded rapidly providing hope that a cure for this lethal disease will be found. One of the major contributions of the field of cell biology to the understanding of malignant diseases has been the identification of growth factors and their receptors as major promoters of transformation and malignant progression. In particular, the appreciation of the central role that receptor tyrosine kinases (RTK) play in different cancers has led to development of effective therapeutic reagents. One of the RTK implicated in malignant progression is the receptor for the type 1 insulin like growth factor (IGF-IR) that has been identified as a target for anti-cancer treatments. Cancer gene therapy is a rapidly developing modality for cancer therapy. Many gene therapy strategies have been developed generally targeting the genes and proteins involved in cancer initiation and progression. To design a successful gene therapy strategy requires an understanding of the molecular basis of cancer progression and knowledge of human and animal genetics and physiology. In the present work, I have introduced two different strategies to inhibit liver metastases formation using established human and murine cancer cell lines. The first strategy is based on targeting the IGF-IR in tumor cells using an antisense technology (chapter 2). This strategy was also shown to be applicable in cancer gene therapy of glioblastoma growing in the brain (chapter 3). Very interestingly and for the first time, we showed that reduction of IGF-IR expression levels in glioma cells can induce a state of dormancy, providing a unique model to study this clinically important phenomenon. As the second strategy, I designed a novel soluble IGF-IR molecule. I showed that expression of this molecule in tumor cells caused an inhibitioDepuis la “déclaration de la guerre contre le cancer” en 1971 et grâce aux avancées en génétique humaine et technologie moléculaire, le domaine de la biologie du cancer s’est rapidement étendu, fournissant l’espoir qu’un traitement pour cette maladie léthale sera trouvé. Une des contributions majeures du domaine de la biologie cellulaire pour la compréhension des maladies malignes a été la mise en évidence du rôle des facteurs de croissance et de leurs récepteurs dans la transformation et la progression maligne. En particulier, le rôle central que jouent les récepteurs à tyrosine kinase (RTK) dans différents cancers a abouti au développement d’agents thérapeutiques efficaces. Un des RTK impliqué dans la progression maligne est IGF-IR, le récepteur de type 1 du facteur de croissance de l’insuline qui a été identifié comme étant une cible pour des traitements anti-cancéreux. La thérapie génique est en pleine voie de développement pour la thérapie du cancer. De manière générale, de nombreuses stratégies de thérapie ont été développées en ciblant les gènes et protéines impliqués dans l’initiation et la progression du cancer. L’élaboration d’une stratégie de thérapie génique efficace nécessite la compréhension des bases moléculaires de la progression du cancer ainsi que la connaissance de la génétique et la physiologie humaine et animale Dans ce travail, j’ai introduis deux stratégies différentes pour inhiber la formation de métastases du foie en utilisant des lignées cellulaires cancéreuses humaines et murines. La première stratégie est basée sur le ciblage de IGF-IR dans des cellules tumorales en utilisant une stratégie antisens (chapitre 2). Cette stratégie s’est également avérée être appliquable à la thérapie génique du cancer de glioblastome dans le cerveau (chapitre 3). De facon intéressante et ce, pour la première fois, nous avons montré que la diminution de l’exp
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Abdelgany, Amr. „Gene therapy for congenital myasthenic syndromes“. Thesis, University of Oxford, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.441062.
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Salooja, Nina. „Towards gene therapy for haemophilia B“. Thesis, University of Oxford, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.393512.
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Wakefield, Philip M. „Gene therapy for duchenne muscular dystrophy“. Thesis, University of Oxford, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365743.
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Chen, Ming-Jen. „Combination gene therapy for colorectal cancer“. Thesis, University of Birmingham, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.273724.
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Oncolytic virotherapy with the adenovirus mutant dl 1520 in combination with chemotherapy has shown clinical response. Approaches to cancer gene therapy involving delivery of enzymes to activate the prodrugs CB 1954 and 5-FC are currently being tested in clinical trials. We hypothesised that the combination of an adenoviral vector equivalent to dl1520 with activation ofCB 1954or 5-FC and the combination of CB 1954 activation with 5-FU may further improve the antitumour effects for colorectal cancer therapy. The initial in vitro data showed that the combination of dl 1520 with CB 1954 activation or 5-FU (metabolite of 5-FC activation) and the combination of CB 1954 activation with 5-FU led to an additive or synergistic cytotoxicity. Subsequent data showed that the incorporation of Ntr or CD-UPRT genes into replicating oncolytic adenoviruses (ROAds) resulted in enhanced Ntr expression or CD-UPRT activity and augmented cytotoxic effects in tissue culture, surpassing the levels and cytotoxic effects mediated by the corresponding replication-defective vectors. When tested in subcutaneous human colon cancer xenografis, Ntr expression mediated by the ROAd was apparently higher than the level mediated by replication-defective CTLI02. Importantly, the antitumoural efficacy of CB 1954 activation mediated by ROAd is significantly superior to that mediated by CTLI 02 (p = 0.01). The ROAds displayed viral replication and oncolysis in vitro and in vivo and these attributes can contribute to the increased gene expression level and enhanced efficacy. Overall, the data suggested that the use of ROAds improved Ntr or CD-UPRT expression and antitumoural efficacy in the presence of corresponding prodrugs and may have the potential to achieve clinical significance in the treatment for colorectal cancer.
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Liljenfeldt, Lina. „CD40L Gene Therapy for Solid Tumors“. Doctoral thesis, Uppsala universitet, Institutionen för immunologi, genetik och patologi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-222705.
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Adenoviral CD40L gene therapy (AdCD40L) is a strong inducer of anti-tumor immune responses via its activation of dendritic cells (DCs). Activated DCs can in turn activate T cells, which are key players in an efficient anti-tumor response. This thesis includes three papers that focus on different aspects of AdCD40L gene therapy. In the first paper, the infiltration of suppressive CD11b+Gr-1+ cells in orthotopic MB49 bladder tumors was investigated and found to be significantly reduced while activated T cells were increased when the tumors had been treated with local AdCD40L gene therapy. Further, AdCD40L could tilt the cells in the tumor microenvironment in favor of an efficient anti-tumor immunity (M1 macrophages and activated T cells) instead of an immunosuppressive environment (CD11b+Gr-1int/low myeloid cells and M2 macrophages). Immunotherapy combined with chemotherapy has shown promising results, and the second paper investigates the combination of AdCD40L gene therapy together with the chemotherapeutic drug 5-Fluorouracil (5-FU). A synergistic effect of the combination treatment on orthotopic MB49 bladder tumors could be demonstrated. The combination therapy resulted in decreased tumor growth, increased survival and systemic MB49-specific immunity, whereas AdCD40L or 5-FU therapy alone had a poor effect on tumor growth. Efficient AdCD40L therapy is dependent on high transduction efficiency in both cancer cells and cells present in the tumor microenvironment. In an attempt to enhance the transduction efficiency, and thereby the therapeutic efficacy, a modified adenovirus was developed for paper three. This modified Ad5PTDf35(mCD40L) could, in comparison with the unmodified Ad5(mCD40L), demonstrate increased transduction capacity of a variety of murine cells. Further, the ability of antigen presenting cells (APCs) to present antigens to T cells was improved after transduction with Ad5PTDf35(mCD40L).
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Chung-Faye, Guy Allen. „Gene therapy strategies for colorectal cancer“. Thesis, University of Birmingham, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.246708.
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Riley, Steven James. „Improving adenovirus vectors for gene therapy“. Thesis, University of Warwick, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.323303.
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Zweiri, Jehad Ahmed. „Suicide immune gene therapy of cancer“. Thesis, King's College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.270793.
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Azevedo, Ferreira Sónia. „Novel approaches to thalassaemia gene therapy“. Thesis, King's College London (University of London), 2013. https://kclpure.kcl.ac.uk/portal/en/theses/novel-approaches-to-thalassaemia-gene-therapy(117f19f2-b791-44ea-9a79-232ce3e600a6).html.
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The haemoglobinopathies are the most prevalent genetically inherited disorders worldwide. Gene therapy for p-thalassaemia is particularly challenging given the requirement for high p-globin chain production in a lineage-specific manner. Advances in lentiviral vector (LV) technology, together with efforts to incorporate erythroid-specific regulatory elements into gene therapy vectors have yielded some promising results in both in vitro and in vivo studies with a number of clinical trials either underway or planned all using an ex vivo approach for the genetic correction of patient haematopoietic stem cells (HSCs). Despite this, current vectors are still sub-optimal: LV integration into the host-cell genome takes place near or within genes being actively transcribed, carrying a potential chance of causing insertional mutagenesis; long-term silencing of integrated transgenes with effect on reproducibility of expression; genetic correction in later developmental stages or adulthood that fails to avert phenotypic manifestations of the condition. This study forms the basis of a long-term programme to develop novel approaches to thalassaemia gene therapy, namely via an in utero delivery route that in principle could be readily applicable worldwide and thus have a positive global impact in the treatment of the haemoglobinopathies. We started with a comparative study with ubiquitous and tissue specific promoter/enhancer combinations in an integration defective lentiviral vector (ID-LV) configuration to assess their ability to express efficiently. Our results show that tissue specific expression of a human P-globin gene (HBE) regulated cassette from within an ID-LV can be obtained albeit at low levels of per vector copy number. The use of ID-LV for targeting HSC clearly awaits the development of versions of this vector system that are capable of replications and retention. We next performed a comparative analysis of UCOE-based lentiviral vectors in a murine neonatal intravascular delivery model system in order to find the ideal marking vector for further in utero studies, hi this comparative study UCOE-eGFP-WPRE proved to be the vector less prone to positional variegation and silencing effects, showing in reproducible levels of expression. Hence, this vector was chosen for further in vivo studies. Our main objective was to address the possibility of an in utero gene therapy approach for the haemoglobinopathies. This methodology aims to take advantage of the migration of HSCs from the foetal liver to bone marrow during development. Therefore, genetic correction of HSC in foetal liver should in principle provide a life-long cure. We initially embarked on a comparative experiment with a UCOE-eGFP-WPRE LV and its twin vector containing a luciferase reporter gene (UCOE-LUC-WPRE), to determine the optimal time of delivery in a murine in utero delivery model system. Injection via the vitelline vessel at E14 and El6 showed little difference with respect to marking of erythroid precursors. However, there appeared to be a higher level of transduced myeloid cells from injections conducted at El6. Following this, we performed vitelline vessel injection at E14 in wild-type mice with a therapeutic GLOBE-based LV containing an HBB cassette consisting of PLCR elements HS2 and HS3 linked to a mini-HBB gene, and its associated transcription termination region. Analysis of recipient mice 3 months post-injection showed vector presence (average copy number 1.347) and detectable haemoglobin levels in peripheral blood.
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Shan, Haidong. „Neuroprotection gene therapy in retinal degeneration“. Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:30f9b5c3-b9a6-4ad0-a952-6840bd73bbc3.
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Retinal degenerative disease, which includes age-related macular degeneration and retinitis pigmentosa, is the main cause of blindness in developed countries. Degeneration of the retinal pigment epithelium (RPE) and photoreceptor cells through apoptosis is believed to be the main mechanism of cell death. X-linked inhibitor of apoptosis (XIAP) is an endogenous anti-apoptotic protein that mediates its effects through the inhibition of caspases - the proteins regulating the final stages of apoptosis. The neuroprotection of XIAP has been demonstrated in various neurodegenerative models. Retinal gene therapy based on adeno-associated virus (AAV) has recently been proven safe and effective in clinical trials of Leber's congenital amaurosis. However, studies are very limited so far about AAV-mediated XIAP effect on degeneration of the RPE and photoreceptor cells. In this thesis, a comprehensive study of AAV-mediated XIAP was performed in the RPE and photoreceptor degenerative models. First, an oxidative stress model was investigated using H2O2 in a human RPE cell line. Second, AAV2-mediated XIAP conferred marked protection on the RPE cells against H2O2 induced apoptosis. Third, an in vivo analysis using confocal scanning laser ophthalmoscope was applied to the NaIO3 induced retinopathy in two transgenic mice (NRL-GFP and B6TGOPN1LW-EGFP). However, subretinal injection of AAV2-XIAP did not rescue photoreceptor cells in the NaIO3-treated animals. Finally, AAV8-XIAP was tested in a rhodopsin mutant mouse line with retinal degeneration (the Rho-/- B6TGOPN1LW-EGFP mouse) but did not reveal any protection on cone photoreceptors. Overall the work in this thesis indicates a limited protection of AAV-mediated XIAP on the RPE and photoreceptor cells in the degenerative models used. XIAP based gene therapy may be helpful for RPE preservation in atrophic AMD, but it needs further research.
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Allen, Edwin Henry Alexander. „Gene therapy for the ocular surface“. Thesis, Ulster University, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.669690.
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Meesmann's epithelial corneal dystrophy (MEeD), which clinically presents with microcysts that can cause irritation, blurred vision or photophobia, is a genetic disorder caused by dominant-negative mutations in the KRT3 and KRT12 genes. Eradicating the mutant protein or tipping the balance strongly in favour of the wild type allele are viable options for therapeutic intervention. Here we studied two therapeutic approaches for suppression of the mutant KRTl2 allele and have developed, characterised and initiated in vivo testing using two novel KRTl2 mouse models. For a transient therapeutic approach, short interfering RNAs (siRNAs) were designed and proved capable of mutation-specific inhibition of the alleles responsible for two MEeD causative mutations (p.Leu132Pro and p.Arg135Thr; 70-90%) in vitro. No off-target issues were observed and suppression of endogenously expressed p.Leu132Pro was also shown in an ex vivo model. For a more generic, yet potentially permanent therapeutic approach, total KRTl2 was suppressed (~50%) with an siRNA expressed from a short hairpin by targeting a region homologus to both the WT and mutant mRNAs. KRT 12 was replaced with a co-expressed recoded allele made resistant to the siRNA. To further develop these potential therapeutics, two novel mouse models were generated allowing evaluation of gene modulation in vivo. (1) A humanised dominant negative mutant model that expresses K12 p.Leu132Pro revealed major changes to corneal phenotype in homozygous animals. Microcysts were observed and keratin expression patterns disrupted. Additionally, RNAseq analysis highlighted over 1600 dysregulated genes, which could feature other potential therapeutic targets for the treatment of symptomatic MEeD. Heterozygous mice presented with a subtler phenotype. (2) A Krt12 luciferase reporter mouse model was optimised and will facilitate live animal corneal imaging, thus aiding the development of topical siRNA delivery formulations. These mouse models in conjunction with our gene silencing development programme pave the way for in vivo assessment of RNA i-based therapeutics for the cornea.
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Sundaram, V. „Gene therapy for inherited retinal diseases“. Thesis, University College London (University of London), 2014. http://discovery.ucl.ac.uk/1418145/.
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Inherited retinal diseases include a number of disorders which typically affect photoreceptor/retinal pigment epithelial function, and can lead to severe visual impairment. Advances in molecular genetics have allowed the identification of many of the genes responsible for particular conditions, and progress in viral gene transfer technology has enabled the replacement of specific genes into the retina. The first human clinical trial of gene therapy for inherited retinal disease was carried out at Moorfields Eye Hospital and UCL Institute of Ophthalmology, involving 12 patients with RPE65 deficiency - an early-onset retinal dystrophy. The results from this trial are described and provide evidence for the safe administration of viral vectors in the eye, and also demonstrate improvements in retinal function in a number of patients. However, the extent and duration of the response did not match that observed in prior animal studies, suggesting improvements in gene expression level may be required in humans. In addition, consideration for future involvement of the foveal region is highlighted, since retinal thinning was observed in a number of patients following subretinal delivery. Achromatopsia is an inherited disorder of congenitally absent cone photoreceptor function. Gene replacement therapy in animal models of achromatopsia has shown evidence of restored cone function, suggesting that this condition may be an appropriate target for gene therapy in humans. Recent studies have suggested that achromatopsia is a progressive condition with deterioration in cone structure with age, implying that the window of opportunity for therapeutic intervention may be narrow. In this study of retinal structure and function (in preparation for a gene therapy trial) in 40 patients with achromatopsia, an age-associated deterioration in cone structure was not identified, suggesting that the age range for potential intervention is wider than recently suggested, and prospective patients should be assessed on an individual basis, irrespective of age.
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Eriksson, Jonas. „Gene therapy tools: oligonucleotides and peptides“. Doctoral thesis, Stockholms universitet, Institutionen för neurokemi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-132271.
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Genetic mutations can cause a wide range of diseases, e.g. cancer. Gene therapy has the potential to alleviate or even cure these diseases. One of the many gene therapies developed so far is RNA-cleaving deoxyribozymes, short DNA oligonucleotides that specifically bind to and cleave RNA. Since the development of these synthetic catalytic oligonucleotides, the main way of determining their cleavage kinetics has been through the use of a laborious and error prone gel assay to quantify substrate and product at different time-points. We have developed two new methods for this purpose. The first one includes a fluorescent intercalating dye, PicoGreen, which has an increased fluorescence upon binding double-stranded oligonucleotides; during the course of the reaction the fluorescence intensity will decrease as the RNA is cleaved and dissociates from the deoxyribozyme. A second method was developed based on the common denominator of all nucleases, each cleavage event exposes a single phosphate of the oligonucleotide phosphate backbone; the exposed phosphate can simultaneously be released by a phosphatase and directly quantified by a fluorescent phosphate sensor. This method allows for multiple turnover kinetics of diverse types of nucleases, including deoxyribozymes and protein nucleases. The main challenge of gene therapy is often the delivery into the cell. To bypass cellular defenses researchers have used a vast number of methods; one of these are cell-penetrating peptides which can be either covalently coupled to or non-covalently complexed with a cargo to deliver it into a cell. To further evolve cell-penetrating peptides and understand how they work we developed an assay to be able to quickly screen different conditions in a high-throughput manner. A luciferase up- and downregulation experiment was used together with a reduction of the experimental time by 1 day, upscaling from 24- to 96-well plates and the cost was reduced by 95% compared to commercially available assays. In the last paper we evaluated if cell-penetrating peptides could be used to improve the uptake of an LNA oligonucleotide mimic of GRN163L, a telomerase-inhibiting oligonucleotide. The combination of cell-penetrating peptides and our mimic oligonucleotide lead to an IC50 more than 20 times lower than that of GRN163L.
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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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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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Brown, Iain. „Gene therapy for sporadic ovarian cancer“. Thesis, University of Aberdeen, 2000. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU602008.
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Ovarian cancer accounts for more deaths than all other gynaecological cancers taken together. The 5 year survival rate can be as high as 80% for cases diagnosed early, but the asymptomatic nature of the disease means that it is most frequently detected in the later stages. By this time, disease has invariably spread beyond the ovaries and the survival rate drops to around 30%. Treatment of ovarian cancer often fails due to a high rate of chemoresistance and novel methods of treatment and detection are required to increase the survival chances of patients. This study sought to determine whether gene therapy for sporadic ovarian cancer could offer a novel and more successful treatment option for the disease. Mutation or abnormal expression of the p53 gene has already been shown to be the most common genetic even in ovarian cancer, being involved in up to 70% of cases. Wild-type p53 was delivered, using liposomes, into p53 mutant ovarian cancer cell lines and this resulted in a restoration of the wild-type functions of the gene, namely cell cycle arrest and apoptosis. The results from the cell line studies suggested that restoration of the wild-type p53 function limit or reduce tumour progression and increase the sensitivity of the tumour to chemotherapy. A mouse model of human peritoneal ovarian cancer was then constructed and the wild-type p53 gene was administered in liposomes into the peritoneum. The results suggested that p53 gene therapy prevents tumours from growing in the mice, when compared to a control gene. It is now known that p53 gene therapy for humans is being clinically assessed. There are a proportion of tumours that do not harbour an abnormal p53 gene, raising the possibility that other tumour suppressor gene mutations may play a role in the molecular genetic control of growth arrest and apoptosis. P53-dependent, apoptosis-regulating family members bcl-2 and bax were analysed immunohistochemically to determine their involvement in ovarian cancer. Both proteins were significantly associated with malignancy and also with overall length of survival, but not associated with the various prognostic factors such as stage and differentiation of tumour. It is unlikely that these genes will become targets for gene therapy in ovarian cancer. Mutation, deletion and hypermethylation of the p53-independent pi6 gene, alter its function, resulting in loss of G1 cell cycle arrest control. The status of methylation of the pi 6 promoter in ovarian tumours was determined and combined with mutation data, resulting in the conclusion that abnormal pi 6 was not a common event in ovarian cancer and is therefore not a likely candidate for gene therapy. This study has contributed to the evergrowing wealth of knowledge on the molecular genetic events of ovarian cancer, and has shown that gene therapy for sporadic ovarian cancer as a clinical application is feasible.
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Demetriades, Anna-Maria. „Periocular gene therapy for ocular angiogenesis“. Thesis, University of Oxford, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.559857.
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Protein delivery to the eye remains an important area in the clinical arena as new therapeutic proteins are being discovered. The focus of this thesis was to establish periocular gene therapy as a novel way of providing localised and sustained release of endogenous anti-angiogenic proteins to the eye. Three endogenous anti-angiogenic proteins of different sizes with established anti-angiogenic activity were chosen for the purpose of our study. The soluble extracellular domain of the vascular endothelial growth factor (VEGF) receptor-1 (sFlt-l; 78kDa), pigment epithelium-derived factor (PEDF; 50kDa) and transforming growth factor beta-I (TGF-B1; 25kDa). In the first part of the thesis, an adenoviral (Ad) vector expressing the reporter gene LacZ was delivered via periocular injection and resulted in infection of extraocular muscles, orbital fat, connective tissues surrounding the eye, and the episclera. Periocular injections of AdsFlt-l, AdPEDF, and AdTGF-B1 were subsequently administered to murine eyes and increased protein levels of sFt-1, PEDF and TGF-B1 were demonstrated in the retina. Thus providing proof-of-principle that a periocular injection of Ad-vectored anti- angiogenic proteins resulted in increased protein levels in the retina and could be a potential method of delivering therapeutic proteins to the posterior segment of the eye. In the second part of the thesis, single periocular inj ections of AdsFlt-l, AdPEDF and AdTGF-J31 were tested in murine models of choroidal neovascularisation, retinal neovascularisation and macular oedema. These studies demonstrated that both a periocular injection of AdsFlt-1 and AdPEDF resulted in significant inhibition of choroidal neovascularisation in an established laser-induced model. However, a periocular injection of AdTGF-B1 had no effect in this chosen model. In addition, a periocular injection of neither AdsFlt-l nor AdPEDF nor AdTGF-B1 resulted in inhibition of retinal neovascularisation. This may have been due to insufficient, non-therapeutic, levels of expressed protein in the retina. A periocular inj ection of AdsFlt-1 resulted in inhibition ofVEGF-induced breakdown of the blood-retinal barrier providing the first proof-of-principle for gene therapy for macular oedema. Interestingly, a periocular injection of AdTGF-J31 resulted in significant inhibition of eye size and development in the neonatal mouse suggesting the involvement of TGF-B1 in postnatal developmental eye growth. In the third part of the thesis, the abili ty to readminister vectors was determined as this would be critical for the application of gene therapy for chronic diseases if a short-acting vector such as Ad was used as the chosen carrier. Repeated periocular delivery of AdPEDF resulted in increased levels ofPEDF in the sclera but not in the choroid and retina. Additional studies are needed to explore the feasibility of repeated injections of Ad vectors including improvement in our understanding of the T cell mediated and humoral immune response in this setting. These studies provide proof-of-principle that periocular delivery of Ad vectors expressing anti-angiogenic transgenes may be a useful approach in the treatment of posterior segment diseases. Further studies are needed to optimize this approach for long-term delivery for the treatment of chronic diseases such as age-related macular degeneration and diabetic retinopathy. With careful consideration, this therapeutic strategy has the potential to be translated into the clinical arena.
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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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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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Shaw, Paul Andrew. „Improving gene delivery for gene therapy and DNA vaccination applications“. Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.614094.
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水野, 正明, 純. 吉田, Masaaki Mizuno und Jun Yoshida. „脳腫瘍の遺伝子治療“. 日本脳神経外科コングレス, 2003. http://hdl.handle.net/2237/10863.
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Thanaketpaisarn, Oranuch. „Optimization of nonviral gene delivery system for in vivo gene therapy“. 京都大学 (Kyoto University), 2005. http://hdl.handle.net/2433/144625.
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Wang, Wen-Hua 1965. „Cytokine gene expression and gene therapy in experimental corneal graft rejection“. Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=38529.
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It has been proposed that CD4+ T cells and cell-mediated immunity play a central role in corneal allograft rejection. Two subsets of CD4+ T cells, Th1 and Th2 cells, are known to cross-regulate each other through their cytokine pattern and the immune response might be directed predominantly in one or the other direction. As such, it has been hypothesized here that predominant Th1 type immune response could lead to corneal allograft rejection, and that previous inflamed corneal beds (high-risk eyes) might augment the Th1 response and thus accelerate the graft rejection.Reverse transcription of mRNA followed by polymerase chain reaction amplification was used to determine the relative gene expression in ocular tissues (cornea and iris/ciliary body) obtained from syngeneic grafts, low- and high-risk allografts. Compared with the syngeneic grafts, mRNA analysis of the low- and high-risk allografts showed a significantly decreasing expression pattern for the Th3 cytokine TGF-beta2, an early peak followed by a decline in the Th2 cytokines IL-4 and IL-10 expression, and a progressively increasing expression of the Th1 cytokines IL-2 and IFN-gamma and the proinflammatory cytokines IL-1beta and TNF-alpha, which paralleled the course of graft rejection. Prevascularization of the recipient eye (high-risk) significantly accelerated the rejection of corneal allografts and the mRNA levels of the Th1 cytokines IL-2 and IFN-gamma and proinflammatory cytokines IL-1beta and TNF-alpha in high-risk allografts were significantly higher and peaked faster than that in low-risk allografts.
In vivo gene transfer using plasmid DNA encoding cytokines is an attractive alternative to modulate the Th1 inflammatory reaction and immune response. This has led to the hypothesis that transferring the gene encoding Th2 cytokine IL-10 into the recipient could prevent or reduce the subsequent corneal allograft rejection through the suppression of Th1-mediated alloimmune response.
Intramuscular injection with in vivo electroporation of IL-10 plasmid DNA was administered at one week before and at one week after corneal transplantation. Corneal allograft survival was significantly prolonged and the rejection rate was significantly reduced after gene therapy with IL-10 plasmid DNA, compared with that in control groups treated with the empty plasmid vector. In IL-10 treated rats, the mRNA expression for the Th1 cytokines IL-2 and IFN-gamma was depressed, and the IL-10 mRNA expression was significantly increased. However, graft survival was not permanent. (Abstract shortened by UMI.)