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1
Aryamvally, Anjali. "Mitochondrial Replacement Therapy: Genetic Counselors’ Experiences, Knowledge and Opinions." University of Cincinnati / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1583998248123854.
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Blechacz, Boris Roman Alexander. "Genetic approaches to the therapy of hepatocellular carcinoma." Thesis, Open University, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.505358.
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Hepatocellular carcinoma (HCC) is a devastating malignancy originating from hepatocytes. There is an urgent need for novel therapeutic approaches. Currently explored gene therapy systems have not yet achieved significant survival benefits. The aim of this thesis was the development and evaluation of novel genetic approaches to this malignancy.
3
Ganly, Ian. "E1B attenuated adenoviruses in genetic therapy for cancer." Thesis, University of Glasgow, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.266588.
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Wood, David Rowe Ding Jiahuan. "Design, optimization, and evaluation of conditionally active gene therapy vectors." Waco, Tex. : Baylor University, 2008. http://hdl.handle.net/2104/5153.
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Choudhury, Sourav Roy. "Developing an Adeno-Associated Viral Vector (AAV) Toolbox for CNS Gene Therapy: A Dissertation." eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/809.
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Neurological disorders – disorders of the brain, spine and associated nerves – are a leading contributor to global disease burden with a sizable economic cost. Adeno-associated viral (AAV) vectors have emerged as an effective platform for CNS gene therapy and have shown early promise in clinical trials. These trials involve direct infusion into brain parenchyma, an approach that may be suboptimal for treatment of neurodegenerative disorders, which often involve more than a single structure in the CNS. However, overall neuronal transduction efficiency of vectors derived from naturally occurring AAV capsids after systemic administration is relatively low. We have developed novel capsids AAV-AS and AAV-B1 that lead to widespread gene delivery throughout the brain and spinal cord, particularly to neuronal populations. Both transduce the adult mouse brain >10-fold more efficiently than the clinical gold standard AAV9 upon intravascular infusion, with gene transfer to multiple neuronal sub-populations. These vectors are also capable of neuronal transduction in a normal cat. We have demonstrated the efficacy of AAV-AS in the context of Huntington's disease by knocking down huntingtin mRNA 33-50% after a single intravenous injection, which is better than what can be achieved by AAV9 at the particular dose. AAVB1 additionally transduces muscle, beta cells, pulmonary alveoli and retinal vasculature at high efficiency, and has reduced sensitivity to neutralizing antibodies in human sera. Generation of this vector toolbox represents a major step towards gaining genetic access to the entire CNS, and provides a platform to develop new gene therapies for neurodegenerative disorders.
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Choudhury, Sourav Roy. "Developing an Adeno-Associated Viral Vector (AAV) Toolbox for CNS Gene Therapy: A Dissertation." eScholarship@UMMS, 2001. http://escholarship.umassmed.edu/gsbs_diss/809.
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Neurological disorders – disorders of the brain, spine and associated nerves – are a leading contributor to global disease burden with a sizable economic cost. Adeno-associated viral (AAV) vectors have emerged as an effective platform for CNS gene therapy and have shown early promise in clinical trials. These trials involve direct infusion into brain parenchyma, an approach that may be suboptimal for treatment of neurodegenerative disorders, which often involve more than a single structure in the CNS. However, overall neuronal transduction efficiency of vectors derived from naturally occurring AAV capsids after systemic administration is relatively low. We have developed novel capsids AAV-AS and AAV-B1 that lead to widespread gene delivery throughout the brain and spinal cord, particularly to neuronal populations. Both transduce the adult mouse brain >10-fold more efficiently than the clinical gold standard AAV9 upon intravascular infusion, with gene transfer to multiple neuronal sub-populations. These vectors are also capable of neuronal transduction in a normal cat. We have demonstrated the efficacy of AAV-AS in the context of Huntington's disease by knocking down huntingtin mRNA 33-50% after a single intravenous injection, which is better than what can be achieved by AAV9 at the particular dose. AAVB1 additionally transduces muscle, beta cells, pulmonary alveoli and retinal vasculature at high efficiency, and has reduced sensitivity to neutralizing antibodies in human sera. Generation of this vector toolbox represents a major step towards gaining genetic access to the entire CNS, and provides a platform to develop new gene therapies for neurodegenerative disorders.
7
Massimo, Gianmichele <1985>. "Hypertension, hypercholesterolemia, hyperaldosteronism: a genetic perspective for personalized therapy." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amsdottorato.unibo.it/6473/1/PhD_Thesis_Gianmichele_Massimo.pdf.
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Essential, primary, or idiopathic hypertension is defined as high BP in which secondary causes such as renovascular disease, renal failure, pheochromocytoma, hyperaldosteronism, or other causes of secondary hypertension are not present. Essential hypertension accounts for 80-90% of all cases of hypertension; it is a heterogeneous disorder, with different patients having different causal factors that may lead to high BP. Life-style, diet, race, physical activity, smoke, cultural level, environmental factors, age, sex and genetic characteristics play a key role in the increasing risk.
Conversely to the essential hypertension, secondary hypertension is often associated with the presence of other pathological conditions such as dyslipidaemia, hypercholesterolemia, diabetes mellitus, obesity and primary aldosteronism. Amongst them, primary aldosteronism represents one of the most common cause of secondary hypertension, with a prevalence of 5-15% depending on the severity of blood pressure. Besides high blood pressure values, a principal feature of primary aldosteronism is the hypersecretion of mineralcorticoid hormone, aldosterone, in a manner that is fairly autonomous of the renin-angiotensin system. Primary aldosteronism is a heterogeneous pathology that may be divided essentially in two groups, idiopathic and familial form.
Despite all this knowledge, there are so many hypertensive cases that cannot be explained. These individuals apparently seem to be healthy, but they have a great risk to develop CVD. The lack of known risk factors makes difficult their classification in a scale of risk. Over the last three decades a good help has been given by the pharmacogenetics/pharmacogenomics, a new area of the traditional pharmacology that try to explain and find correlations between genetic variation, (rare variations, SNPs, mutations), and the risk to develop a particular disease.
8
Massimo, Gianmichele <1985>. "Hypertension, hypercholesterolemia, hyperaldosteronism: a genetic perspective for personalized therapy." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amsdottorato.unibo.it/6473/.
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Essential, primary, or idiopathic hypertension is defined as high BP in which secondary causes such as renovascular disease, renal failure, pheochromocytoma, hyperaldosteronism, or other causes of secondary hypertension are not present. Essential hypertension accounts for 80-90% of all cases of hypertension; it is a heterogeneous disorder, with different patients having different causal factors that may lead to high BP. Life-style, diet, race, physical activity, smoke, cultural level, environmental factors, age, sex and genetic characteristics play a key role in the increasing risk.
Conversely to the essential hypertension, secondary hypertension is often associated with the presence of other pathological conditions such as dyslipidaemia, hypercholesterolemia, diabetes mellitus, obesity and primary aldosteronism. Amongst them, primary aldosteronism represents one of the most common cause of secondary hypertension, with a prevalence of 5-15% depending on the severity of blood pressure. Besides high blood pressure values, a principal feature of primary aldosteronism is the hypersecretion of mineralcorticoid hormone, aldosterone, in a manner that is fairly autonomous of the renin-angiotensin system. Primary aldosteronism is a heterogeneous pathology that may be divided essentially in two groups, idiopathic and familial form.
Despite all this knowledge, there are so many hypertensive cases that cannot be explained. These individuals apparently seem to be healthy, but they have a great risk to develop CVD. The lack of known risk factors makes difficult their classification in a scale of risk. Over the last three decades a good help has been given by the pharmacogenetics/pharmacogenomics, a new area of the traditional pharmacology that try to explain and find correlations between genetic variation, (rare variations, SNPs, mutations), and the risk to develop a particular disease.
9
Holder, Kristina Kichler. "Dynamics of adaptive evolution in two experimental viral systems." Access restricted to users with UT Austin EID Full text (PDF) from UMI/Dissertation Abstracts International, 2001. http://wwwlib.umi.com/cr/utexas/fullcit?p3037499.
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Fuller, Maria. "A gene transfer system derived from human immunodeficiency virus type 1 (HIV-1)." Title page, table of contents, list of abbreviations and epitome only, 2001. http://web4.library.adelaide.edu.au/theses/09PH/09phf9669.pdf.
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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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Ayaz, Serife. "Development Of A Genetic Material Transfer Approach For Gene Therapy." Master's thesis, METU, 2005. http://etd.lib.metu.edu.tr/upload/3/12605939/index.pdf.
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This thesis is focused on the development of a gene delivery system, especially for the purpose of DNA vaccination. DNA expression vectors have the potential to be useful therapeutics for a wide variety of applications. A carrier system was designed to realize the delivery of genes to cells and the promotion of controlled adequate expression in the target cells. The low gene delivery efficiency observed with systems composed of polyplexes is mainly due to low stability of polycation e.g polyethylenimine-DNA complexes and inability of most of the complexes to the reach nucleus after entering the cells. The encapsulation of polyethylenimine-DNA complexes inside the alginate microspheres was expected to provide protection from nuclease-based attack, thereby, increasing the stability of the complex and also to achieve controlled release of the complex at the target tissue.
In this study, controlled release of complexes from alginate microspheres was studied with DNA staining. In Tris-HCl buffer, the release of PEI-DNA complexes were completed in 48 h, however in cell culture medium (DMEM) 18 % of complexes were released in 48 h because of presence of Ca+2 ions in DMEM. Also, in order to provide mucosal gene delivery for mucosal immunization polyethylene glycol (PEG) was introduced into the composition of microspheres and the two systems were compared in terms of release kinetics of the complexes. In the presence of PEG, release of PEI-DNA complexes from alginate microspheres in the cell culture medium (DMEM) were enhanced and 50 % of PEI-DNA were released from the microspheres in 48 h. To understand the effect of the PEG on the surface of microspheres zeta potential analysis and microscopic examination were carried out. By increasing percentage of PEG (0, 15, 30, 50) in microspheres, less negative zeta potential value were measured. Mucoadhesion of alginate and PEG-alginate microspheres were evaluated by using modified microbalance method, and in the presence of PEG enhancement of mucoadhesion was observed. In this way a gene delivery system with a possible route through mucosa of tissues was prepared.
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Hedberg, Rickard. "Preimplantation genetic diagnosis and therapy in humans- Opportunities and risks." Thesis, Örebro universitet, Institutionen för medicinska vetenskaper, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:oru:diva-81532.
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IntroductionPreimplantation Genetic Diagnosis (PGD) was developed in the 1990s and has been used since to diagnose and discard embryos with genetic conditions or chromosomal abnormalities. CRISPR-Cas9 was discovered in 2012 and has been used in research, but has not become clinical practice on humans yet. CRISPR-Cas9 could potentially be applied to treat and prevent genetic disorders.AimThe aim was to investigate the ethical dilemmas of each method through a set of research questions. The ethics of applying PGD according to Swedish guidelines and applying CRISPR-Cas9 on humans was investigated.MethodologyThis was not a systematic literature review. Instead, articles have been selected based on their explanation of each method and uniqueness or volume of ethical arguments surrounding each method, that is of relevance for the discussed issues.ResultsArguments in favour of PGD addressed among other things the somatic and psychological health of future children and parents along with the economical benefits. Arguments against PGD addressed different dilemmas of discarding an embryo and thereby a future individual. Arguments against CRISPR-Cas9 addressed technical limitations, our limited knowledge of genetics and more. Arguments in favour addressed benefits in clinical medicine and research.ConclusionsPGD according to Swedish guidelines was found to be ethically acceptable, since its restrictive use that have not given room for ethically dubious applications. CRISPR-Cas9 was found not to be safe enough for human applications at this moment due to technical limitations. If these were to be solved, caution and restraint must be urged.
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Ahmed, Seemin Seher. "rAAV-Mediated Gene Transfer For Study of Pathological Mechanisms and Therapeutic Intervention in Canavan's Disease: A Dissertation." eScholarship@UMMS, 2014. https://escholarship.umassmed.edu/gsbs_diss/749.
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Canavan’s Disease is a fatal Central Nervous System disorder caused by genetic defects in the enzyme – aspartoacylase and currently has no effective treatment options. We report additional phenotypes in a stringent preclinical aspartoacylase knockout mouse model. Using this model, we developed a gene therapy strategy with intravenous injections of the aspartoacylase gene packaged in recombinant adeno associated viruses (rAAVs). We first investigated the CNS gene transfer abilities of rAAV vectors that can cross the blood-brain-barrier in neonatal and adult mice and subsequently used different rAAV serotypes such as rAAV9, rAAVrh.8 and rAAVrh.10 for gene replacement therapy. A single intravenous injection rescued lethality, extended survival and corrected several disease phenotypes including motor dysfunctions. For the first time we demonstrated the existence of a therapeutic time window in the mouse model. In order to limit off-target effects of viral delivery we employed a synthetic strategy using microRNA mediated posttranscriptional detargeting to restrict rAAV expression in the CNS. We followed up with another approach to limit peripheral tissue distribution. Strikingly, we demonstrate that intracerebroventricular administration of a 50-fold lower vectors dose can rescue lethality and extend survival but not motor functions. We also study the contributions of several peripheral tissues in a primarily CNS disorder and examine several molecular attributes behind pathogenesis of Canavan’s disease using primary neural cell cultures. In summary, this thesis describes the potential of novel rAAV-mediated gene replacement therapy in Canavan’s disease and the use of rAAVs as a tool to tease out its pathological mechanism.
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Ahmed, Seemin Seher. "rAAV-Mediated Gene Transfer For Study of Pathological Mechanisms and Therapeutic Intervention in Canavan's Disease: A Dissertation." eScholarship@UMMS, 2012. http://escholarship.umassmed.edu/gsbs_diss/749.
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Canavan’s Disease is a fatal Central Nervous System disorder caused by genetic defects in the enzyme – aspartoacylase and currently has no effective treatment options. We report additional phenotypes in a stringent preclinical aspartoacylase knockout mouse model. Using this model, we developed a gene therapy strategy with intravenous injections of the aspartoacylase gene packaged in recombinant adeno associated viruses (rAAVs). We first investigated the CNS gene transfer abilities of rAAV vectors that can cross the blood-brain-barrier in neonatal and adult mice and subsequently used different rAAV serotypes such as rAAV9, rAAVrh.8 and rAAVrh.10 for gene replacement therapy. A single intravenous injection rescued lethality, extended survival and corrected several disease phenotypes including motor dysfunctions. For the first time we demonstrated the existence of a therapeutic time window in the mouse model. In order to limit off-target effects of viral delivery we employed a synthetic strategy using microRNA mediated posttranscriptional detargeting to restrict rAAV expression in the CNS. We followed up with another approach to limit peripheral tissue distribution. Strikingly, we demonstrate that intracerebroventricular administration of a 50-fold lower vectors dose can rescue lethality and extend survival but not motor functions. We also study the contributions of several peripheral tissues in a primarily CNS disorder and examine several molecular attributes behind pathogenesis of Canavan’s disease using primary neural cell cultures. In summary, this thesis describes the potential of novel rAAV-mediated gene replacement therapy in Canavan’s disease and the use of rAAVs as a tool to tease out its pathological mechanism.
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Firrman, Jenni Ann. "ENHANCEMENT OF hFVIII ACTIVITY THROUGH LC MODIFICATIONS FOR GENE THERAPY OF HEMOPHILIA A." Diss., Temple University Libraries, 2015. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/335863.
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Microbiology and ImmunologyPh.D.
Gene therapy for Hemophilia A (HA) using the recombinant Adeno-associated virus (rAAV) offers an alternative to classic treatment, which consists of FVIII protein infusions. However, due to limitations associated with rAAV and the FVIII protein itself, the end result is a transgene expression below therapeutic limits. One approach to improving the therapeutic value of rAAV gene therapy for HA is to engineer a more active FVIII protein through genetic modifications. Preliminary testing revealed that canine FVIII Light Chain (kLC) enhances coagulation activity, and that it would be possible to improve FVIII activity through modifications of the light chain. Through the process of engineering, evaluation, and negative selection of kLC, a final construct was engineered. The hLC-K12 is a human Light Chain (hLC) construct containing 12 amino acid changes that work together to enhance coagulation activity. A comparison of the FVIII clotting activity to the amount of protein produced determined that hLC-K12 produced a 3.28 fold increase in specific activity over hLC in vitro. Similar in vitro results were observed when hLC-k12 was tested with the X5 heavy chain (X5HC), a heavy chain that has been genetically modified to enhance production. CD4KO/HA mice were injected with a rAAV vector carrying the hLC-K12 gene in conjugation with a rAAV vector carrying the X5HC gene. Replacing the hLC vector with the hLC-K12 vector produced an average 7.43 fold increase in FVIII clotting activity. An ELISA assay revealed no significant difference between productions of the heavy or light chains at any time point. By comparing the clotting activity to the amount of protein produced, it was determined that the increase in coagulation activity was due to an increase in specific activity. In fact, replacing the hLC vector with the hLC-K12 vector resulted in an average 5.8 fold increase in FVIII specific activity. The K12 modifications were evaluated using a single chain FVIII conformation. In vitro, the addition of the K12 mutations to the human heavy chain, hHCK12BDD, resulted in a 4.3 fold increase in clotting activity, but no increase in protein production. There was however, a 3.3 fold increase in specific activity of the protein. Adding the K12 mutations to the X5 heavy chain, X5K12BDD, in vitro, resulted in a 2.7 fold increase in clotting activity and a 1.42 fold increase in specific activity of the protein. Single chain rAAV vectors were packaged and delivered to CD4KO/HA mice. Compared to mice injected with hFVIIIBDD, the hHCK12BDD produced an average 4.6 fold increase in clotting activity. An ELISA revealed no significant difference in production between these two groups. However, mice injected with hHCK12BDD produced FVIII with an average of 4.13 fold increase in specific activity. Similarly, when compared to mice injected with X5FVIIIBDD, the X5K12BDD produced an average 2.14 fold increase in clotting activity. An ELISA assay demonstrated no significant increase in protein production between these two groups. However, when compared to X5BDD, mice injected with the X5K12BDD vector produced FVIII with an average 1.98 fold increase in specific activity. Results demonstrate that the K12 light chain modifications are able to enhance clotting activity of hFVIII both in vitro and in vivo, using either a dual chain or single chain delivery method. In order to determine the mechanism of enhancement, hFVIIIBDD and hHCK12BDD protein was partially purified and tested for activity. Results demonstrated that the hHCK12BDD protein produced a specific activity of 39,153.69 Units/mg, which is a 6.28 fold increase over hFVIIIBDD specific activity, which was 6,237.92 Units/mg. Measurement of conversion from FX to FXa revealed that the hHCK12BDD protein generated a higher amount of FXa at a quicker rate. In conclusion, these results provide evidence that the K12 modifications enhance specific activity through an increase in FXa generation.
Temple University--Theses
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BASSANINI, GIULIA. "EFFECT OF DIET THERAPY ON GUT MICROBIOME IN RARE GENETIC DISEASES." Doctoral thesis, Università degli Studi di Milano, 2021. http://hdl.handle.net/2434/828416.
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In the last decades, several studies have explored the human microbiota in different body niches, notably in the gastrointestinal tract. The gut microbiota has been recognized as an additional organ that co-evolves with humans and interacts with host physiology. The gut microbiota plays a key role in the host metabolism and the extraction of energy from food.
Diet is the strongest factor shaping the gut microbiota: differences in macronutrient intake may select the growth of specific microbial taxa. Indeed,
the diet provides different substrates such as undigested carbohydrates for fermentation, affecting the composition of the gut microbiota and,
consequently, the production of microbial metabolites. Short chain fatty acids (SCFAs), mainly acetate, propionate, and butyrate, are the major end products of anaerobic fermentation by gut microbes.
Modifications in the proportions of the gut microbes and, consequently, the production of SCFAs have been suggested to play a role in several pathological conditions. If the diet is modifiable to stimulate the growth of beneficial microbes, it could represent a target for prevention and treatment of
a possible dysbiosis.
In congenital metabolic disorders such as phenylketonuria (PKU) and glycogen storage disease (GSD), diet is considered the mainstay for the treatment of the pathology. PKU diet is based on the restriction of phenylalanine intake and the supplementation of essential amino acids and micronutrients. PKU diet is similar to a vegan alimentation since all the animal products are excluded. GSD dietary management provides the intake of slow-release carbohydrates to prevent hypoglycaemia and limits the assumption of simple sugars.
In this research, we aimed to analyze the impact of dietary therapy on the composition of the gut microbiota and the production of SCFAs in two congenital metabolic disorders (PKU and GSD). PKU and GSD diseases showed an alteration of the gut microbiome. If diet represents the only treatment of the disease and is not modifiable, a personalized intervention to restore a healthy gut microbiome is to consider. Nowadays, the attention is focused on the supplementation of probiotics and prebiotics.
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Kodimattam, Joseph George. "Molding a Better Humanity? Ethical Implications of Human Genetic Modifications for Enhancement." Thesis, Linköping University, Centre for Applied Ethics, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-12266.
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The study analyzes the ethical implications of human gene transfer technology for enhancement. Although human gene transfer technology is widely accepted on therapeutic grounds the non-therapeutic use of gene transfer technology remains to be a gray zone for moral deliberation. The present discussion addresses several ethical issues concerning the impacts of human gene transfer technology on individuals, the society, and future people. Accordingly, the study examines major ethical issues concerning the use of human gene transfer technology in general and genetic enhancement in particular, and reliability of the putative demarcation between therapy and enhancement, and further proposes ethical guidelines for non-therapeutic application of human gene transfer technology. A special attention is given to three major ethical issues, such as our obligation to future generations, problems concerning justice, fairness, and equality, and the problem of uncertainty.
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Ross, Colin J. D. "Immuno-isolation gene therapy for lysosomal storage disease /." *McMaster only, 2001.
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Taivassalo, Tanja. "Exercise training as therapy for mitochondrial myopathies : physiological, biochemical and genetic effects." Thesis, McGill University, 2000. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=37845.
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Patients with mitochondrial myopathies characteristically exhibit pronounced exercise intolerance, often associated with lactic acidosis, tachycardia and muscle weakness. These clinical features are attributable to impaired electron transport chain function in skeletal muscle. The usual etiology is a primary defect in mitochondrial DNA (mtDNA), where the severity of impairment is presumably linked to the ratio of mutant to wild-type mtDNA. This dissertation presents novel therapeutic approaches to these genetic defects, aimed at attenuating mitochondrial dysfunction and ameliorating the clinical condition by employing exercise training alone or in conjunction with pharmacological therapy. Dichloroacetate (DCA) was administered to augment mitochondrial capacity by activating pyruvate dehydrogenase, thereby decreasing lactic acidosis. Endurance and resistance training paradigms were employed to induce mitochondrial and satellite cell proliferation respectively. The goals were to augment respiratory chain function, increase levels of wild type mtDNA, and reverse effects of chronic inactivity. The effects of these treatments on functional and mitochondrial capacity were defined by changes in: (1) work capacity, oxygen utilization, and circulatory responses during maximal exercise; (2) heart rate and blood lactate during submaximal exercise; (3) recovery kinetics of phosphate-containing metabolites measured using phosphorus magnetic resonance spectroscopy ( 31P MRS); (4) scores on a quality of life questionnaire. The cellular correlates for these indices were defined by changes in: (1) mitochondrial volume, (2) respiratory chain enzyme activity, and (3) levels of mutant/wild-type mtDNA. Although DCA administration alone lowered blood lactate, endurance training was more effective in improving exercise capacity, heart rate and blood lactate, 31P MRS recovery kinetics, and quality of life. Increased mitochondrial volume and respiratory chain function were closely linked
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Zech, M. H. "Genetic control of MTOR to improve adoptive T cell therapy of tumours." Thesis, University College London (University of London), 2014. http://discovery.ucl.ac.uk/1420953/.
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Adoptive T cell therapy to treat cancer in combination with re-directing specificity through T cell receptor (TCR) gene transfer, represents an effective therapeutic option. However, reduced effector responses due to the immunosuppressive tumour microenvironment and insufficient long-term engraftment of transferred cells represent two potential limitations. Tumours often employ mechanisms to inhibit T cell responses including secretion of TGFβ and depleting the tumour microenvironment of amino acids. The main aim of this PhD project was to develop a strategy to enhance T cell function for tumour therapy. The mammalian target of rapamycin (mTOR) pathway regulates CD8 T cell differentiation such that high mTOR activation leads to enhanced effector whilst low mTOR activation leads to increased T cell memory formation. Two retrovirus constructs have been designed whereby one expresses the positive mTOR regulator Rheb and the other expresses the negative mTOR regulator Pras40. Rheb transduction into CD8 T cells resulted in enhanced activation of mTOR, increased effector functions and partial resistance to TGFβ and low arginine concentrations. Pras40 overexpression led to a decrease in the activation of mTOR and reduced effector functions. Rheb transduced CD8 T cells expanded efficiently upon antigen encounter in vivo, followed by pronounced T cell contraction. Pras40 transduced T cells were unable to expand in vivo, but persisted at low numbers and acquired a central memory phenotype. Tumour bearing mice treated with TCR re-directed CD8 T cells transduced with Rheb showed improved tumour protection. Pras40 overexpression resulted in the loss of the protective function of TCR re-directed T cells. Together, the data show that gene transfer can be used to regulate mTOR activity in T cells. Enhancing mTOR activity led to improved tumour control despite reducing memory formation. Permanent mTOR inhibition, on the other hand, preserved some memory characteristcs of T cells but deteriorated their tumour protective functions.
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Tang, Yizhe. "Modification of adenovirus capsid proteins for gene therapy applications." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2009. https://www.mhsl.uab.edu/dt/2010r/tang.pdf.
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Aints, Alar. "Vector development for suicide gene therapy /." Stockholm, 2002. http://diss.kib.ki.se/2002/91-7349-199-3.
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Krishna, Delfi. "Investigation of the role of target cell factors in retrovirus transduction." Diss., Available online, Georgia Institute of Technology, 2005, 2005. http://etd.gatech.edu/theses/available/etd-11212005-102548/.
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Thesis (Ph. D.)--Chemical and Biomolecular Engineering, Georgia Institute of Technology, 2006.Harish Radhakrishna, Committee Member ; Mark Prausnitz, Committee Co-Chair ; Joseph Le Doux, Committee Chair ; Timothy Wick, Committee Member ; Richard Compans, Committee Member ; Athanassios Sambanis, Committee Member.
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Thakur, Sanjay, and n/a. "The ethics of preimplantation genetic diagnosis." University of Otago. Department of Philosophy, 2006. http://adt.otago.ac.nz./public/adt-NZDU20060816.105106.
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Preimplantation genetic diagnosis is a technique used in the field of assisted reproduction. The technique is applied to embryos that have been created in vitro, in order to facilitate the selection of embryos according to particular genetic parameters. The use of preimplantation genetic diagnosis by prospective parents at high risk for having a child affected by a genetic disorder has facilitated the birth of unaffected children. Preimplantation genetic diagnosis has already been used for other purposes, such as screening for gender, and could in principle be used to screen for a wide range of genetic traits. The aim of this thesis is to provide good answers to the ethical questions provoked by the advent and continuing development of preimplantation genetic diagnosis.
The thesis is divided into four parts. Part One provides a brief overview of the science of genetic selection. Part Two is centred on a discussion of two ethical principles. The principle of procreative liberty is based upon the idea that acts of interference in the reproductive lives of others should be avoided unless there is good justification for such acts. The principle of procreative beneficence is based upon the idea that prospective parents should select the child, of the possible children they could have, who is expected to have the best life. I will argue that the principle of procreative liberty should be applied to acts of interference in individuals� freedom to use preimplantation genetic diagnosis, while the principle of procreative beneficence should be applied to acts of selecting children.
In Part Three, I will endorse a position that accords embryos a relatively low moral status, reject the arguments of the disability rights critique, argue that the eugenic aspects of preimplantation genetic diagnosis do not warrant much concern, and develop a framework for critically evaluating slippery slope arguments. Finally, in Part Four, specific applications of preimplantation genetic diagnosis will be examined in detail. Although each application raises unique ethical questions, this thesis aims to demonstrate that the consistent application of the principles and preliminary conclusions developed in Parts Two and Three provides the best means for determining how PGD should be used and which uses should be restricted.
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Lindahl, Katarina. "Osteogenesis Imperfecta : Genetic and Therapeutic Studies." Doctoral thesis, Uppsala universitet, Metabola bensjukdomar, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-208942.
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Osteogenesis imperfecta (OI) is a heterogeneous disease of connective tissue, the cardinal symptom being fractures and severity ranging from mild to lethal. Dominant mutations in collagen I, encoded by COL1A1 and COL1A2, cause >90% of cases. To delineate genotype-phenotype correlations and pharmaco-genetic response, collagen I was sequenced in 150 unrelated Swedish families and clinical data were collected in Paper I. Mutation type, gene affected, and N- to C-terminal location correlated with phenotype and severity. Bisphosphonate response assessed by calculated yearly change in lumbar spine bone mineral density (BMD) was inversely related to age and BMD at treatment initiation. Mutations associated with a more severe phenotype exhibited an increased response after 2 years; however, all types of OI responded well. To investigate the effect of naturally occurring variations in collagen I, the only common coding single nucleotide polymorphism (rs42524 in COL1A2) was genotyped in 2004 healthy men in Paper II. Heterozygous genotype was associated with decreased BMD and an increased risk of stroke. An adolescent with repeated fractures despite a markedly high BMD harbored a unique C-terminal procollagen cleavage-site mutation in COL1A1, which motivated extensive investigations in concert with a similar COL1A2 case in Paper III. The probands were found to have impaired procollagen processing, incorporation of collagen with retained C-propeptide in matrix and increased mineral to matrix ratio, which demonstrates that C-propeptide cleavage is crucial to normal bone mineralization and structure. Bisphosphonate therapy has insufficient effect in OI, and as classical OI is a dominant disorder severe cases would benefit from silencing of the mutated allele. In Paper IV and V small interfering RNAs (siRNAs) were used to allele-specifically target primary human bone cells heterozygous for I) a coding polymorphism in COL1A2 and II) insertion/deletions in the 3’UTR of COL1A1 and COL1A2. Results were promising with altered allele ratios and decreased mRNA levels in the predicted fashion. To summarize, this thesis found that collagen I is crucial to bone and connective tissue and that collagen I mutations create markedly diverse phenotypes. Age, BMD and pharmaco-genetic effects influence the response to bisphosphonate therapy in individuals with OI; however, novel approaches are needed. Utilizing allele-specific siRNAs may be a way forward in the treatment of severe OI.
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Roncato, Rossana. "Innovative strategies for tailoring therapy in cancer patient pharmacogenetics and therapy personalization in metastatic colorectal cancer patients treated with irinotecan." Doctoral thesis, Università degli studi di Padova, 2017. http://hdl.handle.net/11577/3425853.
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Pharmacogenetics focuses on inter-subject variation in drug therapeutic effects and toxicity depending on genetic polymorphisms. Irinotecan and fluoropyrimidine, currently used in cancer chemotherapy, are characterized by a sometimes unpredictably severe toxicity. Pharmacogenomics was largely applied in the last years to the irinotecan-based colorectal cancer (CRC) treatment personalization with limited data regarding validated marker of severe toxicity.
In the first part of my thesis, I have been focusing on the investigation of innovative pharmacogenetic markers of neutropenia or gastrointestinal toxicity irinotecan-related using the “tagging polymorphisms (SNPs)” (TagSNPs) approach. Since therapeutic implications of cancer-related inflammation have gained great attention in recent years, innovative prospects for the optimization of tailored therapy arose. Two hundred and fifty metastatic CRC patients, homogeneously treated with an irinotecan-including regimen (FOLFIRI), have been collected retrospectively for this study. Clinical parameters of toxicity (by NCI-CTC scale) and response to the therapy (by WHO criteria) were monitored all along the study. They were genotyped for 246 htSNPs characterizing 22 transcriptional regulators and cytokines inflammation-related genes; positive findings were replicated in a cohort of 167 metastatic CRC patients receiving FOLFIRI-based therapy. One polymorphism (rs1053004) in STAT-3 gene resulted predictive of severe GI toxicity in both discovery and replication cohort with a protective effect toward the risk of developing grade 3-4 events (OR=0.51 CI=0.27-0.99 p=0.045; OR=0.38 CI=0.15-0.95 p=0.038, respectively). Additional variants in NRs genes, especially HNF4α and VDR, although not validated, were suggested to contribute to determining the risk of developing neutropenia and GI toxicity. Preliminary pharmacokinetic data supported the observed genotype/phenotype clinical associations. A validated contribution of STAT-3 rs1053004 in determining GI toxicity risk after FOLFIRI therapy was pointed out. Further potential predictive markers of irinotecan-related toxicity were suggested. These findings could represent a further step towards personalized FOLFIRI therapy.
UGT1A1*28 polymorphism has been demonstrated in the last years to have an impact on irinotecan pharmacokinetics and toxicity to the treatment. Although, the adoption of a pre-emptive UGT1A1*28 genotyping to increase irinotecan safety and to better characterize patient “Diagnosis Related Groups”, for therapy reimbursement purposes in clinical practice, is still limited. The second part of my thesis aimed to estimate the effect of UGT1A1*28 on the costs associated with irinotecan-related toxicity. A retrospective analysis of the costs of toxicity management was conducted on a subset of the aforementioned population of 250 mCRC patients. 243 mCRC patients treated with FOLFIRI have been genotyped for UGT1A1*28. The mean predicted cost per patient was higher for *1/*28 (1,119€) and *28/*28 (4,886€), as compared to *1/*1 (812€) (P<0.001). This is consistent with a different grade 4 toxicity profile among the three groups of patients, and a higher frequency of costly interventions like hospitalization among patients with the *28 allele.
The aim of the third part of my thesis consisted of evaluating the implementation of the routine application of prospective DPYD risk variants and UGT1A1*28 screening at the National Cancer Center CRO of Aviano. A Pharmacogenetic implementation infrastructure has been set-up starting from January 2014 for the prevention of irinotecan (UGT1A1*28 rs8175347) and/or fluoropyrimidine (DPYD rs3918290, rs55886062, rs67376798)-associated toxicity in the clinical routine of the National Cancer Center CRO of Aviano. Genotyping was performed by PCR-based methods, such as pyrosequencing, Sanger sequencing, and fragment analysis. A digital Pharmacogenomic report including the dose-adjustment recommended according to the published pharmacogenetics guidelines will finally be embedded in patients’ clinical record and ultimately made available to the medical personnel. From September 2011 to September 2016, a total of 393 patients were genotyped for such variants at CRO-Aviano. Three hundred and eighty-six out of 393 patients were screened for at least one DPYD variants and 40 for UGT1A1*28. Of these patients, 9 patients (2.58%) were found to carry at least one DPYD variants, and two patients (5.00%) were found to carry two *28 risk alleles for UGT1A1. Moreover, twenty-three patients out of 393 (5.85%) were referred for toxicity from the CRO-Aviano oncologists.
In conclusion, in this work of thesis, interesting molecular markers with a predictive value on pharmacokinetics and pharmacodynamics of irinotecan were described. A possible application of these parameters in the clinical practice will be useful to design a tailored irinotecan dosing based on peculiar characteristics of each patient. In addition to the prevention of severe toxicity, pre-treatment UGT1A1*28 genotyping should be considered to save economic resources related to the management of irinotecan-related toxicities and for innovative reimbursement strategies. Plus, the implementation of pre-emptive pharmacogenetics tests is now part of a European Project (U-PGx) with the aim of providing the final proof of pharmacogenetics efficacy in increasing drug safety when fully integrated into the clinical practice.I pazienti oncologici non sempre rispondono analogamente al medesimo trattamento farmacologico ed è il motivo per cui la somministrazione della stessa dose di un farmaco chemioterapico in una popolazione omogenea di pazienti può implicare la manifestazione di differenti risposte e tossicità. Tale variabilità intersoggettiva può essere determinata da interazioni complesse fra componenti fisiologiche, ambientali e fattori genetici individuali. Per valutare la correlazione tra genotipo e fenotipo si rendono necessari studi retrospettivi e prospettici per stabilire se alcuni polimorfismi (SNP) possano essere marcatori di tossicità od efficacia. Una volta stabilita la validità clinica di tali marcatori, la loro implementazione nella clinica può risultare un processo altrettanto ostico e articolato, così come la definizione della rimborsabilità di tale test genetico all’interno della Sanità Pubblica Italiana. La presente tesi di dottorato descrive la mia attività di ricerca che si è diversificata in vari aspetti della personalizzazione della terapia nel paziente oncologico durante i tre anni di dottorato, in particolar modo focalizzandosi sul tumore al colon retto (CRC) metastatico. Nella prima parte della tesi mi sono occupata di investigare l’esistenza di innovativi marker farmacogenetici predittivi di neutropenia e tossicità gastrointestinale (GI) utilizzando l’approccio del “tagging polymorphisms (SNP)” (TagSNPs). Essendo ben riconosciuto il ruolo dell’infiammazione nello sviluppo del tumore, negli ultimi anni si sono aperte innovative prospettive di ottimizzazione della terapia in questo campo. Dallo studio di 250 pazienti caucasici omogeneamente trattati con FOLFIRI in prima linea per CRC metastatico sono stati genotipizzati 246 htSNPs in 22 geni regolatori della trascrizione e citochine legate all’infiammazione. Un polimorfismo nel gene di STAT-3 è risultato predittivo di tossicità GI severa sia nella coorte esplorativa sia in quella di validazione con un effetto protettivo nei confronti degli effetti tossici di grado 3 e 4. A seguire, mi sono occupata della valutazione dell’effettiva utilità clinica del test genetico per UGT1A1*28 misurando l'effetto di tale polimorfismo sui costi associati alla tossicità da irinotecano all’interno dell’Istituto CRO di Aviano essendone già ampliamente riconosciuta la validità clinica. Su un sottogruppo di 243 pazienti della casistica descritta nel paragrafo precedente, è stata condotta un'analisi retrospettiva dei costi di gestione della tossicità in relazione al genotipo UGT1A1*28. Il costo medio previsto per paziente si è rivelato essere superiore per pazienti con genotipo *1/*28 (1.119 €), e *28/*28 (4.886 €), rispetto a quelli con genotipo *1/*1 (812 €) (p <0.001). Nell’ultima parte di questa tesi ho descritto il mio contributo nella messa a punto dei processi per istituire all’interno della struttura ospedaliera del CRO di Aviano il test preventivo di alcuni riconosciuti marcatori genetici di tossicità in principalmente due geni: DPYD (rs3918290, rs55886062, rs67376798) e UGT1A1*28 (r8175347) per tutti i pazienti oncologici eleggibili a trattamento con fluoropirimidine o irinotecano. Le peculiarità dell’infrastruttura e le sue normative sono state prese in considerazione per una efficace integrazione del test genetico pre-trattamento nel flusso di lavoro clinico dell’ospedale. Il processo di diagnostica farmacogenetica inizia con una prescrizione del test farmacogenetico in formato digitale da parte dell’oncologo, successivamente personale medico formato si occupa di effettuare il prelievo e di farlo pervenire presso l’unità di Farmacologia Sperimentale e Clinica, dove personale tecnico altrettanto specializzato effettuerà l’analisi genetica e produrrà un referto seguendo un protocollo approvato ISO-9001. Il referto si costituisce di due parti: una tecnica e una clinica. Successivamente alla firma in digitale del personale tecnico per la validazione dell’analisi genetica effettuata, la firma digitale da parte di personale clinico assieme ad una interpretazione dei dati viene apportata. L’interpretazione dei dati sarà effettuata sulla base delle raccomandazioni di dosaggio farmacogenetiche nazionali ed internazionali disponibili. La realizzazione di test di farmacogenetica preventivi è ora parte di un progetto europeo (U-PGx), che ha come obiettivo ultimo quello di fornire la prova definitiva dell’efficacia dell’approccio farmacogenetico quando completamente integrato nella pratica clinica.
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Donnelly, Louise. "Genetic and clinical determinants of lipid-lowering response to statin therapy in diabetes." Thesis, University of Dundee, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.505632.
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Bick, Alexander George. "At the Heart of the Genome: Rare Genetic Variation, Cardiovascular Disease, and Therapy." Thesis, Harvard University, 2014. http://dissertations.umi.com/gsas.harvard:11399.
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Studies of large families with inherited single gene disorders identified a role of rare genetic variation as a cause of disease and enabled gene-based diagnosis. The increasing availability of population-scale genomic sequencing implies the potential to extend gene-based diagnosis from individuals with monogenic disease to the prediction of disease risk in the general population. Cardiovascular disease (CVD), as a highly heritable condition with significant public health burden, represents an excellent place to consider the promise and limitations of extending our understanding of rare variation in single gene disorders to the general population.
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Cherry, Shannon Marie. "Speech Pathologists and Knowledge Regarding Communication Disorders with Genetic Inheritance." University of Cincinnati / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1212085523.
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Heller, Raoul. "Engineering of human artificial mini-chromosomes." Thesis, University of Oxford, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.360317.
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Pritchard, Lynn Edward. "The genetics of type 1 (insulin-dependent) diabetes mellitus." Thesis, University of Oxford, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.241656.
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Brandén, Lars J. "The development of synthetic gene delivery systems /." Stockholm, 2001.
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Guhasarkar, Dwijit. "A Walk on the Fine Line Between Reward and Risk: AAV-IFNβ Gene Therapy for Glioblastoma: A Dissertation." eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/843.
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Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. The current standard-of-care treatment including surgery, radiation and temozolomide (TMZ) chemotherapy does not prolong the survival satisfactorily. Here we have tested the feasibility, efficacy and safety of a potential gene therapy approach using AAV as gene delivery vehicle for treatment of GBM.
Interferon-beta (IFNβ) is a cytokine molecule also having pleiotropic anticancerous properties. Previously it has been shown by our group that AAV mediated local (intracranial) gene delivery of human IFNβ (hIFNβ) could be an effective treatment for non-invasive human glioblastoma (U87) in orthotopic xenograft mouse model.But as one of the major challenges to treat GBM effectively in clinics is its highly invasive property, in the current study we first sought to test the efficacy of our therapeutic model in a highly invasive human GBM (GBM8) xenograft mouse model.
One major limitation of using the xenograft mouse model is that these mice are immune-compromised. Moreover, as IFNβ does not interact with cross-species receptors, the influence of immune systems on GBM remains largely untested. Therefore to test the therapeutic approach in an immune-competent mouse model, we next treated a syngeneic mouse GBM model (GL261) in an immune-competent mouse (C57B6) with the gene encoding the species-matched IFNβ (mIFNβ). We also tested if combination of this IFNβ gene therapy with the current standard chemotherapeutic drug (TMZ) is more effective than any one of the therapeutic modes alone. Finally, we tested the long term safety of the AAV-mIFNβ local gene therapy in healthy C57B6 mice.
Next, we hypothesized that global genetic engineering of brain cells expressing secretory therapeutic protein like hIFNβ could be more beneficial for treatment of invasive, migratory and distal multifocal GBM. We tested this hypothesis using systemic delivery of AAV9 vectors encoding hIFNβ gene for treatment of GBM8 tumor in nude mice.
Using in vivo bioluminescence imaging of tumor associated firefly luciferase activity, long term survival assay and histological analysis of the brains we have shown that local treatment of AAV-hIFNβ for highly invasive human GBM8 is therapeutically beneficial at an early growth phase of tumor. However, systemic delivery route treatment is far superior for treating multifocal distal GBM8 tumors. Nonetheless, for both delivery routes, treatment efficacy is significantly reduced when treated at a later growth phase of the tumor.
In syngeneic GL261 tumor model study, we show that local AAV-mIFNβ gene therapy alone or in combination with TMZ treatment can provide significant survival benefit over control or only TMZ treatment, respectively. However, the animals eventually succumb to the tumor. Safety study in the healthy animals shows significant body weight loss in some treatment groups, whereas one group shows long term survival without any weight loss or any noticeable changes in the external appearances. However, histological analysis indicates marked demyelinating neurotoxic effects upon long term exposures to mIFNβ over-expressions in brain. Overall, we conclude from this study that AAV-IFNβ gene therapy has great therapeutic potential for GBM treatment in future, but the therapeutic window is small and long term continuous expression could have severe deleterious effects on health.
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Guhasarkar, Dwijit. "A Walk on the Fine Line Between Reward and Risk: AAV-IFNβ Gene Therapy for Glioblastoma: A Dissertation." eScholarship@UMMS, 2007. http://escholarship.umassmed.edu/gsbs_diss/843.
Full textAbstract:
Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. The current standard-of-care treatment including surgery, radiation and temozolomide (TMZ) chemotherapy does not prolong the survival satisfactorily. Here we have tested the feasibility, efficacy and safety of a potential gene therapy approach using AAV as gene delivery vehicle for treatment of GBM.
Interferon-beta (IFNβ) is a cytokine molecule also having pleiotropic anticancerous properties. Previously it has been shown by our group that AAV mediated local (intracranial) gene delivery of human IFNβ (hIFNβ) could be an effective treatment for non-invasive human glioblastoma (U87) in orthotopic xenograft mouse model.But as one of the major challenges to treat GBM effectively in clinics is its highly invasive property, in the current study we first sought to test the efficacy of our therapeutic model in a highly invasive human GBM (GBM8) xenograft mouse model.
One major limitation of using the xenograft mouse model is that these mice are immune-compromised. Moreover, as IFNβ does not interact with cross-species receptors, the influence of immune systems on GBM remains largely untested. Therefore to test the therapeutic approach in an immune-competent mouse model, we next treated a syngeneic mouse GBM model (GL261) in an immune-competent mouse (C57B6) with the gene encoding the species-matched IFNβ (mIFNβ). We also tested if combination of this IFNβ gene therapy with the current standard chemotherapeutic drug (TMZ) is more effective than any one of the therapeutic modes alone. Finally, we tested the long term safety of the AAV-mIFNβ local gene therapy in healthy C57B6 mice.
Next, we hypothesized that global genetic engineering of brain cells expressing secretory therapeutic protein like hIFNβ could be more beneficial for treatment of invasive, migratory and distal multifocal GBM. We tested this hypothesis using systemic delivery of AAV9 vectors encoding hIFNβ gene for treatment of GBM8 tumor in nude mice.
Using in vivo bioluminescence imaging of tumor associated firefly luciferase activity, long term survival assay and histological analysis of the brains we have shown that local treatment of AAV-hIFNβ for highly invasive human GBM8 is therapeutically beneficial at an early growth phase of tumor. However, systemic delivery route treatment is far superior for treating multifocal distal GBM8 tumors. Nonetheless, for both delivery routes, treatment efficacy is significantly reduced when treated at a later growth phase of the tumor.
In syngeneic GL261 tumor model study, we show that local AAV-mIFNβ gene therapy alone or in combination with TMZ treatment can provide significant survival benefit over control or only TMZ treatment, respectively. However, the animals eventually succumb to the tumor. Safety study in the healthy animals shows significant body weight loss in some treatment groups, whereas one group shows long term survival without any weight loss or any noticeable changes in the external appearances. However, histological analysis indicates marked demyelinating neurotoxic effects upon long term exposures to mIFNβ over-expressions in brain. Overall, we conclude from this study that AAV-IFNβ gene therapy has great therapeutic potential for GBM treatment in future, but the therapeutic window is small and long term continuous expression could have severe deleterious effects on health.
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Ayuso, López Eduard. "Genetic manipulation of the pancreas: cell and gene therapy approaches for type 1 diabetes." Doctoral thesis, Universitat Autònoma de Barcelona, 2006. http://hdl.handle.net/10803/3557.
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La diabetes de tipo 1 resulta de la destrucción autoinmune de las células ß pancreáticas, que conduce a una falta en la producción de insulina y la consiguiente hiperglucemia. La terapia sustitutiva con inyecciones subcutáneas de insulina permite a los pacientes llevar un vida activa, sin embargo esta terapia es imperfecta y no evita la aparición de graves complicaciones secundarias. El transplante de páncreas o islotes pancreáticos se ha realizado con éxito en algunos pacientes, sin embargo la escasez de donantes impide que esta terapia se pueda aplicar a todos los individuos diabéticos. Por ello, una gran cantidad de esfuerzos se han centrado en la diferenciación de células madre, embrionarias o adultas, en células ß. Las células de la médula ósea (BMC) poseen propiedades de célula madre adulta y además son fáciles de obtener, por ello se han propuesto como una fuente alternativa para la formación de nuevas células ß. El factor de crecimiento a la insulina de tipo I (IGF-I) participa en la regeneración muscular e incrementa la atracción y diferenciación de BMC en el músculo dañado. Además, la expresión de IGF-I específicamente en células ß de ratones diabéticos es capaz de regenerar la masa de células ß. Así, el primer objetivo de este trabajo fue estudiar la capacidad de la expresión de IGF-I en las células ß para atraer y diferenciar las BMC en nuevas células ß, tanto en ratones sanos como en ratones diabéticos. Con esta finalidad se transplantó la médula ósea de ratones transgénicos que expresaban la proteína verde fluorescente (GFP) constitutivamente, en los ratones transgénicos para IGF-I. Los resultados obtenidos demostraron que ni la sobreexpresión de IGF-I en células ß, ni la inducción de diabetes mediante estreptozotocina fueron causa suficiente para atraer y diferenciar las BMC en células ß pancreáticas in vivo. Estos datos sugerían que la regeneración del páncreas endocrino observada en los ratones transgénicos para IGF-I no era mediada por las BMC, indicando que la replicación de células ß preexistentes o bien la diferenciación a partir de precursores no hematopoyéticos son los mecanismos que actuarían en la regeneración de las células ß mediada por IGF-I.La diabetes se ha intentando curar mediante estrategias de terapia génica, sin embargo hasta el momento no se ha conseguido ninguna terapia efectiva. La recuperación completa del paciente diabético de tipo 1 requeriría la regeneración de las células ß. Una aproximación para conseguir este objetivo es la manipulación genética del páncreas endocrino in vivo, con la finalidad de expresar factores que induzcan replicación o neogénesis de las células ß y además contrarrestar la respuesta inmune. Sin embargo, el riesgo de inducir pancreatitis al manipular el páncreas es elevado, y por tanto se han realizado escasos intentos de modificar genéticamente este órgano hasta la fecha. Por ello, nuevas aproximaciones de transferencia génica in vivo son necesarias para avanzar en el desarrollo de nuevas aproximaciones de terapia génica para la diabetes. En este trabajo hemos estudiado la eficiencia de diferentes vectores virales y diferentes vías de administración para transducir el páncreas in vivo, tanto en ratones como en perros. En primer lugar, observamos que las células ß pancreáticas fueron transducidas eficientemente por adenovirus inyectados vía sistémica en ratones a los cuales se les había cerrado la circulación hepática. Este resultado obtenido con vectores adenovirales de primera generación también se obtuvo cuando usamos vectores adenovirales de última generación, también llamados gutless. Además de vectores adenovirales, también se estudio la capacidad de transducir el páncreas de los vectores adenoasociados de serotipo 8 (AAV8). Así, se demostró que la vía de administración de los vectores AAV8 por el conducto pancreático era más efectiva que la administración de estos vectores por vía endovenosa o intraperitoneal.
El páncreas del perro presenta una estructura lobular y una vascularización similar al humano, por tanto constituye un buen modelo para ensayar estrategias de transferencia génica a páncreas. En este trabajo se estudió la capacidad de los vectores adenovirales para transferir genes a páncreas in vivo en animales sometidos a un clamp circulatorio de los vasos pancreáticos. Adenovirus con el gen marcador de la ß-galactosidasa se inyectaron en la vena pancreaticoduodenal y el clamp se mantuvo durante 10 minutos. Usando esta técnica se consiguió transducir células acinares, ductales y también islotes pancreáticos sin evidencias de daño pancreático. Esta técnica también se ensayó con éxito en un perro diabético.
Por consiguiente, la metodología descrita en este trabajo puede ser usada para transducir el páncreas in vivo, ya sea en ratones o en perros, con la finalidad de estudiar la biología de las células ß o bien para desarrollar nuevas aproximaciones terapéuticas para la diabetes mellitus y otras enfermedades pancreáticas.
Type 1 diabetes is characterized by progressive destruction of pancreatic ?-cells, resulting in insulin deficiency and hyperglycemia. Insulin replacement therapy allows diabetic patients to lead active lives, but this therapy is imperfect and does not prevent development of severe secondary complications. Transplantation of pancreatic tissue or islets has been performed successfully in a limited numbers of patients. However, the shortage of donors is a primary obstacle that prevents this treatment from becoming more widespread. Therefore, many efforts have been focused on differentiating embryonic or adult stem cells into ß-cells. Bone marrow cells (BMCs) are an important source of easily procurable adult stem cells and have been proposed as an alternative source of ß-cells. Insulin-like growth factor-I (IGF-I) participates in skeletal muscle regeneration and enhances the recruitment of BMCs at the sites of muscle injury. In addition, IGF-I expression in ß-cells of diabetic transgenic mice regenerates pancreatic ß-cell mass. Therefore one of the objectives of this study was to investigate whether IGF-I expression in ß-cells could increase BMC recruitment and differentiation into ß-cells under steady-state conditions or after STZ treatment. To this end, BMCs from ß-actin/GFP transgenic donor mice were transplanted into IGF-I transgenic mice. Our experiments have demonstrated that IGF-I overexpression or STZ-induced pancreatic damage were not sufficient to recruit and differentiate GFP-labelled BMCs into ß-cells in vivo, indicating that these cells did not contribute to the endocrine pancreas regeneration observed in IGF-I transgenic mice. These data suggest that replication of pre-existing ß-cells and/or differentiation from non-BMC precursors is the most likely mechanism for IGF-I-mediated regeneration.
Diabetes mellitus has long been targeted, as yet unsuccessfully, as being curable with gene therapy. Recovery from type 1 diabetes requires ß-cell regeneration. One approach to do so is by genetically engineering the endocrine pancreas in vivo to express factors that induce ß-cell replication and neogenesis and counteract the immune response. However, the pancreas is difficult to manipulate and pancreatitis is a serious concern, which has made effective gene transfer to this organ elusive. Thus, new approaches for gene delivery to the pancreas in vivo are required. In this study we have examined different viral vectors and routes of administration in rodents and also in large animals, to determine the most efficient method to deliver exogenous genes to the pancreas. First, we observed that pancreatic ß-cells were efficiently transduced to express ß-galactosidase after systemic injection of adenoviral vectors in mice with clamped hepatic circulation. This was true both for first generation as well as for helper-dependent adenoviral vectors. In addition to adenoviruses, we have compared the ability of AAV vectors to transduce the pancreas in vivo after intravascular, intraperitoneal or intraductal delivery, being the last the most efficient route of administration.
Like the human pancreas, the canine pancreas is compact, with similar vascularization and lobular structure. It is therefore a suitable model in which to assess gene transfer strategies. Here we examined the ability of adenoviral vectors to transfer genes into the pancreas of dogs in which pancreatic circulation has been clamped. Adenoviruses carrying the ß-galactosidase (ß-gal) gene were injected into the pancreatic-duodenal vein and the clamp was released 10 min later. These dogs showed ß-gal-positive cells throughout the pancreas, with no evidence of pancreatic damage. ß-gal was expressed mainly in acinar cells, but also in ducts and islets. ß-gal expression in the exocrine pancreas of a diabetic dog was also found to be similar to that observed in healthy dogs.
Thus, the methodology described herein may be used to transfer genes of interest to murine and canine pancreas in vivo, both for the study of islet biology and to develop new gene therapy approaches for diabetes mellitus and other pancreatic disorders.
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Kim, A. Rang M. S. "Discontinuing Enzyme Replacement Therapy in Patients with Lysosomal Storage Diseases due to Significant Clinical Decline." University of Cincinnati / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1396522371.
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Chen, Yan, and 陳岩. "Recombinant adenovirus and adeno-associated virus mediated BMP2 and BMP4 gene therapy for new bone formation." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2003. http://hub.hku.hk/bib/B31244038.
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Parathyras, John Burns. "Molecular genetic analysis of human immunodeficiency virus antiretroviral therapy response in South Africa : a pharmacogenetics study." Thesis, Link to the online version, 2007. http://hdl.handle.net/10019/453.
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Svahn, Mathias G. "DNA analogs for the purpose of gene therapy /." Stockholm, 2007. http://diss.kib.ki.se/2007/978-91-7357-290-3/.
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梁頌偉 and Chung-wai Leung. "Novel gene transfer vector targeted high affinity IL-2 receptor bearing cell." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2001. http://hub.hku.hk/bib/B31226280.
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Lee, Kin-wah Terence, and 李建華. "Targeted gene delivery using a receptor-mediated gene transfer system and chemosensitivity in hepatocellular carcinoma." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2000. http://hub.hku.hk/bib/B3122295X.
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The Best MPhil Thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), Li Ka Shing Prizepublished_or_final_version
Pathology
Master
Master of Philosophy
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Patterson, Sonya Marie. "Development of a cell-specific targeting strategy for therapeutic gene delivery vectors." Thesis, University of Newcastle Upon Tyne, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.364767.
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Leung, Chung-wai. "Novel gene transfer vector targeted high affinity IL-2 receptor bearing cell /." Hong Kong : University of Hong Kong, 2001. http://sunzi.lib.hku.hk/hkuto/record.jsp?B25248698.
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Hakariya, Hayase. "Non-Genetic Cell-Surface Modification with a Self-Assembling Molecular Glue." Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/263577.
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付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム京都大学
新制・課程博士
博士(医科学)
甲第23116号
医科博第127号
京都大学大学院医学研究科医科学専攻
(主査)教授 藤田 恭之, 教授 渡邊 直樹, 教授 岩田 想
学位規則第4条第1項該当
Doctor of Medical Science
Kyoto University
DFAM
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Mapagu, Maria Cristina. "Molecular and Genetic Features Underlying Treatment Response in Relapsed Ovarian Cancer." Thesis, The University of Sydney, 2018. http://hdl.handle.net/2123/20181.
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Most women with advanced epithelial ovarian cancers (EOC) will relapse. Currently, there are no predictive tests to guide clinicians when treating relapsed EOC. The aim of the work described in this thesis was to identify genomic markers that predict response to treatments commonly used in relapsed EOC: platinum chemotherapy, pegylated liposomal doxorubicin (PLD) and endocrine therapy. We identified women treated with platinum, PLD or endocrine therapy, performed molecular tests on primary tumour and associated these with response. In platinum-treated patients, we confirmed that homologous recombination (HR) deficiency, either HR pathway gene mutations or an overall ‘HRD score’, was associated with higher response rates. CCNE1 gain/amplification was associated with non-response to platinum (p=0.03). In PLD treated patients,BRCA mutations were associated with increased response (p=0.04). We examined HGSOC expression subtypes (C1, C2, C4, C5) in relation to platinum and PLD response, and although no significant associations were found there was an over-representation of the C4 subtype in platinum non-responders and an under-representation of the C2 subtype in PLD responders. Using RNA-Seq, we identified 41 differentially expressed genes (DEGs)between PLD-sensitive and -resistant, newly established HGSOC cell lines; 4 of these genes were also differentially expressed in tumours from PLD complete responders and non-responders. In patients treated with endocrine therapy, increasing ERa histoscores in HGSOC tumours may be associated with response (p=0.07). Using RNA-Seq, we identified oestrogen (E2)-regulated genes in ER+ HGSOC cell lines with differential E2growth response. We identified 27 DEGs between HGSOC tumours from endocrine therapy responders and non-responders, and found that most of these genes were E2-regulated. This E2-regulated signature may identify patients with ER+ HGSOC that respond to endocrine therapy. Taken together, we found that molecular markers in primary tumour could potentially predict treatment outcome in relapsed EOC. Our data provide a rationale for testing primary tumour for specific predictive molecular markers. If validated, these predictive markers could be used to personalise treatment of relapsed EOC.
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Iwasenko, Jenna Maree Biotechnology & Biomolecular Sciences Faculty of Science UNSW. "Genetic factors of cytomegalovirus and other herpesviruses that influence outcomes of antiviral therapy in transplantation." Publisher:University of New South Wales. Biotechnology & Biomolecular Sciences, 2009. http://handle.unsw.edu.au/1959.4/43703.
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The clinical impact of human cytomegalovirus (CMV) and progression to CMV disease in immunocompromised patients has been reduced by therapeutic strategies using ganciclovir, valganciclovir, foscarnet and cidofovir. However, extensive antiviral therapy increases the risk of antiviral resistance due to mutations in the UL97 protein kinase and UL54 DNA polymerase. Co-infection with HHV-6 or HHV-7 is also associated with increased CMV reactivation and disease. Genotypic CMV antiviral resistance was identified in 38% of Australian immunocompromised patients. While UL97 mutations only were identified in 23% of patients, additional UL54 mutations, with the potential to confer multidrug resistance, were detected in 15% of patients. Antiviral resistant CMV strains were found to emerge rapidly in highly immunocompromised patients, and some strains were able to persist in the absence of selective pressure. Three new mutations were identified (UL97 - N597D, UL54 - F412S, D485N). N597D was characterised by recombinant phenotyping and conferred minimal ganciclovir resistance. Neither baculovirus nor coupled transcription/translation yielded full-length UL54 protein (pUL54; ~140 kDa) for activity assays. However, truncated pUL54 (~66 kDa) was purified after prokaryotic expression. HHV-6 and HHV-7 co-infection was a common clinical occurrence; with 36% of liver transplant recipients infected with HHV-6 (11% persistent) and 80% with HHV-7 (52% persistent). ValGCV therapy did not significantly alter the incidence of HHV-6, HHV-7 or co-infection. The most prevalent co-infection pattern was CMV, HHV-6 and HHV-7 (46%) and both CMV and HHV-7 (38%). CMV reactivation was predominantly independent of HHV-6/HHV-7, although 27% of patients had initial HHV-7 reactivation. Despite frequent co-infection, HHV-6 and HHV-7 were not associated with clinical disease, with possible exception of HHV-7 and acute cellular rejection. CMV antiviral resistance remains a significant issue in transplantation, emphasising the importance of antiviral resistance testing in an era of widespread prophylaxis. New mutations in UL97 and UL54 continue to be identified. Further characterisation of UL54 mutations using polymerase activity assays would increase our knowledge of enzymological basis of antiviral resistance. Co-infection with HHV-6 and HHV-7 is common in transplant recipients, but does not play a significant role in disease. Similar co-infection rates between valGCV-treated and untreated patients indicate that valGCV is not highly effective against HHV-6 and HHV-7.
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Limberis, Maria. "A lentiviral gene transfer vector for the treatment of cystic fibrosis airway disease." Title page, synopsis and list of contents only, 2002. http://web4.library.adelaide.edu.au/theses/09PH/09phl735.pdf.
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"16th September 2002." Accompanying CD contains 2 MPEG clips with accompanying text, and a copy in PDF format of: Recovery of airway cystic fibrosis transmembrane conductance regulator function in mice with cystic fibrosis after single-dose lentivirus-mediated gene transfer / M. Limberis ... [et al.], published in Human gene therapy vol. 13 (2002). Bibliography: leaves xxix-li. This thesis focuses on modulating the physical barriers of the airway epithelium with mild detergents, so as to enhance gene transfer by a HIV-1 based lentivirus vector in vivo. The efficiency of the gene transfer was evaluated in the nasal airway of C57B1/6 mice using the Lac Z marker gene. This demonstration of lentivirus-mediated in vivo recovery of CFTR function in CF airway epithelium illustrated the potential of combining a pre-conditioning of the airway surface with a simple and brief HIV-1 based gene transfer vector exposure to produce therapeutic gene expression in the intact airway.
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McKechnie, Victoria Margaret. "Variation in the NS5A gene of Hepatitis C Virus in response to interferon alpha therapy." Thesis, University of Glasgow, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.301364.
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Keeler, Allison M. "Gene Therapy for Very Long Chain Acyl-coA Dehydrogenase Deficiency Using Adeno-Associated Virus Vectors: A Dissertation." eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/632.
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Very long chain acyl-coA dehydrogenase (VLCAD) is the rate-limiting step in mitochondrial fatty acid oxidation. VLCAD deficient mice and patients’ clinical symptoms stem from not only an energy deficiency but also long-chain metabolite accumulations. VLCAD deficient mice were treated systemically with 1x10 12 vector genomes of rAAV9-VLCAD. Expression was detected in the liver, heart and muscle. Also substantial expression of VLCAD was noted in the brain, where it was expressed across different sections of the brain and in different cell types with different morphologies. Biochemical correction was observed in vector-treated mice beginning two weeks post-injection, as characterized by a significant drop in long chain fatty acyl accumulates in whole blood after an overnight fast. Changes persisted through the termination point around 20 weeks post injection. Magnetic resonance spectroscopy (MRS) and tandem mass spectrometry (MS/MS) revealed normalization of intramuscular lipids in treated animals. Correction was not observed in liver tissue extracts, but cardiac muscle extracts showed significant reduction of long chain metabolites. Disease-specific phenotypes were characterized, including thermoregulation and maintenance of euglycemia after a fasting cold challenge. Internal body temperatures of untreated VLCAD-/- mice dropped below 20°C and the mice became lethargic, requiring euthanasia. In contrast all rAAV9-treated VLCAD-/- mice and the wild-type controls maintained body temperatures. rAAV9-treated VLCAD-/- mice maintained euglycemia, whereas untreated VLCAD-/- mice suffered hypoglycemia following a fasting cold challenge. These promising results suggest rAAV9 gene therapy as a potential treatment for VLCAD deficiency in humans.