Dissertations / Theses: 'Cystic fibrosis Gene therapy'

1

Davies, Gwyneth. "Outcome measures for cystic fibrosis gene therapy clinical trials." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/28414.

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Background: Cystic fibrosis (CF) is a life-shortening, chronic respiratory disease caused by mutations in the CFTR gene. Novel therapeutic agents such as gene therapy aim to correct CFTR and to demonstrate evidence of molecular, functional and (ultimately) clinical efficacy. It was hypothesised that currently used methods to detect these changes may be optimised to enhance sensitivity and allow quantification, and facilitate an understanding of geographical effects within the airway. Methods: Outcome measures were investigated within two UK CF Gene Therapy Consortium studies; a longitudinal observational study ('Run-In'), and a single dose gene therapy study ('Pilot') which investigated safety and functional efficacy. Measurement of airway function with spirometry and lung clearance index in the Run-In study allowed investigation of variability and change over time and comparison with other outcomes. Development of methodology and data analysis from measurements of potential difference (PD) in the nose and lung in the Pilot study allowed investigation of these as measures of functional efficacy. Results: In the Run-In study, the choice of external reference source was crucial for interpretation of spirometry outcomes. Airway physiology outcomes correlated with structural changes on chest CT however were limited in their ability to detect site of airway abnormality. There was some evidence that disease severity was associated with intra-subject variability and affected rate of change over time. In the Pilot study, airway PD was shown to change post gene therapy within individuals but the responses were not universal and depended on the definitions used. A novel method of nasal PD quantification did not improve an ability to quantify change. Conclusions: There is no single universal outcome measure in CF, but it is important to take account of the patient population in terms of disease severity. Whilst it would be inappropriate to relate PD outcomes with clinical outcomes in the Pilot study; this will be an important relationship to understand in the future in order to allow rational design of CF gene therapy clinical trials.

2

Rose, Andrew C. "Studies on the expression of the murine CFTR gene : implications for gene therapy." Thesis, University of Oxford, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365354.

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Dragomir, Anca. "Approaches to Pharmacological Treatment and Gene Therapy of Cystic Fibrosis." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3845.

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McKay, Tristan Rowntree. "Investigations toward gene therapy for hepatobiliary disease in cystic fibrosis." Thesis, Imperial College London, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.392184.

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5

Cooney, Ashley L. "Integrating viral vectors as a gene therapy approach for cystic fibrosis." Diss., University of Iowa, 2018. https://ir.uiowa.edu/etd/6083.

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Cystic fibrosis (CF) is the most common autosomal recessive genetic disease in Caucasian populations. CF affects multiple organ systems including pancreas, liver, intestines, sweat glands, and male reproductive organs, however the leading cause of morbidity and mortality in CF patients is chronic lung disease. CF is caused by a mutant cystic fibrosis transmembrane conductance regulator (CFTR) gene which leads to chloride (Cl-) and bicarbonate (HCO3-) anion dysregulation at the airway surface. Without adequate anion exchange, thick, viscous mucus accumulates at the airway surface allowing bacterial colonization to occur. Complementing CFTR in the appropriate airway cells restores the anion channel activity in CFTR-deficient cells. The ultimate goal for CF gene therapy is to design an integrating vector that would lead to persistent and efficient expression of CFTR in the airways. Performing gene therapy experiments is dependent upon a relevant animal model. The CF pig is a large animal model similar in size, anatomy, and physiology to humans. Importantly, the CF pig recapitulates human lung disease. From the CF pig, we have learned much about CF lung disease and have developed relevant assays to measure anion channel correction. We have learned that loss of CFTR leads to a decreased airway surface ASL pH, bacterial killing ability, and increased mucus viscosity. Standardized assays have been developed to evaluate the change in current by Ussing chambers, ASL pH, bacterial killing in vivo and ASL pH and viscosity on primary airway cultures in vitro. Ultimately, these metrics allow us to make conclusions about the efficiency of CFTR restoration. Viral vectors are promising candidates for CF gene therapy. Viral vectors such as adenovirus (Ad), adeno-associated virus (AAV), and pseudotyped lentiviral vectors such as feline immunodeficiency virus (FIV) or human immunodeficiency virus (HIV) can efficiently transduce airway cells and express CFTR. Ad and AAV have both been tested in CF clinical trials, but CFTR expression was transient, if detected at all. Understanding vector biology and overcoming barriers in the lung have allowed us to improve vector delivery to the airways. However, the next major hurdle was achieving persistent expression. Ad and AAV are both transiently expressing vectors, and vector readministration is implausible due to the presence of neutralizing antibodies that develop against the vector. Creating a hybrid nonviral/viral vector in which the integrating nonviral piggyBac transposon system is delivered by an Ad or AAV vector has allowed us to achieve persistent expression in mice. In a third integrating vector system, lentiviral vectors have historically been challenging to work with due to low titer levels. However, improvement in vector purification methods have allowed us to validate a lentiviral vector as a viable gene therapy option. In total, we have validated three integrating vector systems by restoring CFTR to CF pigs to correct the phenotypic defect.

6

Scott, Emily Siân. "Improving the efficiency of liposome-mediated gene transfer for cystic fibrosis gene therapy." Thesis, University of Cambridge, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.624332.

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Jannetta, Evelyn Elena. "Qualitative study of cystic fibrosis (CF) patients' expectations of gene therapy." Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/8745.

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Introduction: Gene therapy is currently being developed for people with cystic fibrosis (CF), a life-threatening condition for which there is no cure. The UK CF Gene Therapy Consortium are preparing for a multi-dose gene therapy trial of sufficient duration that clinical benefit may be seen. Aims: The current study aimed to explore the expectations and beliefs of cystic fibrosis (CF) patients involved in the preparatory phase of the gene therapy trial (the Run-in study), from which participants will be selected for the multi-dose actual gene therapy trial. Method: Twelve participants (six with mild and six with moderate CF) were interviewed using a semi-structured interview. Interviews were recorded, transcribed verbatim and then analysed using a Constructivist Grounded Theory approach. Results: Since entering the Run-in study, half of the patients had increased their expectations of gene therapy being an effective future treatment. Most of the participants hoped to derive clinical benefit from the trial itself though half were unsure of what to expect. Whilst half of the participants expressed the hope of a future cure for CF, the remainder saw gene therapy only in terms of an improved treatment. Participants used several strategies to manage their expectations including not thinking too far ahead and trusting the research team. Discussion: The findings indicate that participants in the Run-in trial are generally eager to be involved in the gene therapy trial and have developed a strong sense of trust in the research team conducting the trials. The levels of optimism expressed for personal benefit from trial were higher than those from earlier studies. Some of the positive expectations were unlikely to be met by the gene therapy trial and participants risk disappointment. However other patients participated with apparently realistic expectations and it seems likely that some patients would have participated even without prospect for personal benefit. Possible areas of psychological support are discussed e.g. a standard clinical interview for all those not accepted for the gene therapy trial; screening for anxiety pre-, during and post-participation.

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Jaffe, Adam. "Assessment and feasibility of gene therapy for cystic fibrosis in children." Thesis, Imperial College London, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.589769.

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Middleton, Peter Gordon. "Cystic fibrosis ion transport and the effect of CFTR gene transfer." Thesis, Imperial College London, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.307399.

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Kwilas, Anna R. "Respiratory Syncytial Virus Based Vectors for the Treatment of Cystic Fibrosis." The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1284384649.

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11

Ramachandran, Shyam. "Regulation Of gene expression in cystic fibrosis: implications for biology and therapeutics." Diss., University of Iowa, 2012. https://ir.uiowa.edu/etd/2613.

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Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that when mutated causes the disease cystic fibrosis (CF). Many obstacles hinder the understanding of CF disease pathogenesis, impeding advancements in understanding how mutations cause disease, and slowing the progress towards new treatments. To this end, we have profiled the transcriptome (mRNA and microRNA) of human and newborn pig CF and non-CF airway epithelia. We show that the use of cross-species transcriptomics allows the identification of genes differentially expressed owing to the loss of CFTR, and not due to confounding environmental or secondary disease progression influences. The identification of reduced OAS1 expression in CF samples is a case in point. We also demonstrate the utility of transcriptome profiling and longitudinal studies in pigs, providing greater understanding of the molecular mechanisms underlying CF disease progression. MicroRNAs (miRNAs) comprise a large family of ~21-nt long non-coding RNAs that function as key post-transcriptional regulators of gene expression. Very little is known of how CFTR is regulated in the cell, both transcriptionally and post-transcriptionally. We discovered three miRNAs: miR-509-3p, miR-494 and miR-138 with possible CFTR regulatory functions. miR-509-3p or -494 directly target the CFTR mRNA, and decrease CFTR levels when over expressed; while inhibiting them had the opposite effect. Upon stimulating human airway epithelial cells with TNFα or IL-1β, we observed an increase in expression of both miRNAs mediated in part by the NF-κB transcription factor complex, with a concurrent decrease in CFTR expression. Gene ontology classification of predicted targets of miR-509-3p and/or miR-494 expressed in the airway epithelium revealed enrichment for genes in ion transport pathways. To our knowledge, this is the first suggestion of a possible role for miRNAs regulating a broad range of important epithelial electrolyte and fluid transport proteins. The study of miR-138 mediated regulation of CFTR expression has led to novel discoveries in the field of CFTR transcriptional control. We discovered SIN3A to be a novel transcriptional repressor of CFTR, interacting with CTCF on the CFTR promoter at the -20.9 kb DHS. By validating SIN3A as a conserved target of miR-138, we also discovered miR-138 to be a novel transcriptional regulator/activator of CFTR. The most common CFTR mutation, ΔF508, causes protein misfolding, degradation, and CF. Manipulating the miR-138/SIN3A regulatory network improved the biosynthesis of CFTR-ΔF508, restoring Cl- transport to human CF airway epithelia. To our knowledge, this is the first example of an individual miRNA having such broad regulatory functions. This discovery also provided novel targets for restoring CFTR function in cells affected by the most common CF mutation. To this end, we are utilizing the molecular signatures of miR-138 over-expression and SIN3A knockdown to identify candidate genes for RNA interference screens, and to identify candidate small molecule drugs that might mimic the effects of these two interventions. The goal of this approach is to develop a new therapeutic agent that restores anion transport to airway epithelia and other cell types and tissues affected by CF.

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Bergau, Anna. "Developing vectors for cystic fibrosis gene therapy : improving the longevity and tissue specificity of gene expression." Thesis, Imperial College London, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.424514.

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13

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

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

14

Davies, Lee. "The electrical manipulation of bio-formulations for delivery to the lung." Thesis, University of Oxford, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365799.

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15

Semir, Frappart David de. "Reparación de mutaciones en el gent CFTR como estrategia de terapia génica para la fibrosis quística." Doctoral thesis, Universitat Pompeu Fabra, 2005. http://hdl.handle.net/10803/7084.

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La fibrosis quística (fq) es la enfermedad autosómica recesiva más frecuente en la población caucasoide con una frecuencia de portadores de 1/25. Las manifestaciones clínicas más importantes son las infecciones crónicas recurrentes del pulmón conllevando al deterioro del mismo. En esta tesis nos propusimos corregir dos mutaciones en el gen cftr, responsable de la fq, en la linea celular de epitelio bronquial ib3.1 de genotipo (f508del/w1282x). Para ello, mediante citometría de flujo y microscopía confocal pusimos a punto la incorporación no viral (vectores pei, geneporter y citofectina) de oligonucleótidos modificados (quimeraplastos y oligonucleótidos fosforotioato) y de fragmentos sfhr en estas células. Los quimeraplastos son oligonucléotidos quiméricos de rna y dna y los oligonucleótidos fosforotioato contienen enlaces fosforotioato para evitar su degradación además de enlaces fosfodiéster. Ambos, son capaces de estimular los mecanismos de reparación intrínsecos de las células para corregir a nivel de dna mutaciones puntuales. Los fragmentos sfhr (small fragment homologous replacement) son fragmentos de pcr de longitud variable con la secuencia salvaje del gen que se intercambian con las secuencias genómicas diana mutadas por recombinación homóloga para revertir mutaciones puntuales (cambios de 1 nucleótido, pequeñas inserciones o deleciones). Para estimar el porcentaje de corrección génica adaptamos la técnica diagnóstica de pcr-ola a nuestro modelo para utilizarla como metodología de cuantificación. Además, hemos confirmado que las células ib3.1 tienen los mecanismos de reparación de mutaciones puntuales activos tanto por rt-pcr como por un ensayo in vitro en e.coli con el plásmido pksm4021 que contiene el gen de resistencia a ampicilina y el gen de resistencia a kanamicina inactivado por una mutación puntual. Los resultados que se publicaron en los siguientes artículos nos han permitido extraer las siguientes conclusiones: las células Ib3.1 de epitelio bronquial de fq son competentes en cuanto al sistema de reparación mmr. La incorporación celular de quimeraplastos, oligonucleótidos monocadena y fragmentos sfhr es muy eficiente en las células ib3.1. El lípido catiónico citofectina, el policatión pei y la electroporación, aunque no el lípido catiónico geneporter, son sistemas de transfección eficientes para incorporar oligonucleótidos modificados en el núcleo de las células ib3.1. Los poliplejos de pei y los lipoplejos de citofectina son internalizados por distintos mecanismos aunque en ambos casos los oligonucleótidos modificados son degradados significativamente en las células ib3.1. El análisis genescan de electroferogramas fluorescentes constituye un sistema fiable, fácil, sensible y seguro para evaluar y cuantificar la degradación intracelular y extracelular de oligonucleótidos marcados con fluorescencia en fluidos biológicos. La tecnología de pcr-ola constituye un sistema fiable y preciso de cuantificación de reparación génica aplicable a modelos celulares heterocigotos. Los fragmentos sfhr pueden actuar como cebador artefactual en las reacciones de pcr y generar un artefacto que da lugar a falsos positivos en la detección de conversión génica. Es indispensable diseñar los cebadores de detección de la modificación génica fuera de la región de homología con los fragmentos sfhr. La corrección génica de mutaciones puntuales mediada por quimeraplastos, oligonucleótidos monocadena y fragmentos sfhr es un proceso ineficiente en las células ib3.1. Será necesario estimular los mecanismos endógenos de reparación génica para incrementar la frecuencia de reparación génica hasta niveles terapéuticos en dichas células.

16

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.

17

Walker, Wendilywn E. "Towards gene therapy for cystic fibrosis : enhanced green fluorescent protein as a reporter of promoter activity." Thesis, University of Edinburgh, 2005. http://hdl.handle.net/1842/27597.

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The aim of this project was to investigate the use of the Enhanced Green Fluorescent Protein (EGFP) as a reporter of CFTR promoter activity. six vectors were created coupling portions of the CFTR locus to EGFP in GCVs. Small plasmids were made by conventional cloning procedures, while large PAC vectors were made by a double recombination method employing both homologous and Cre recombinase/loxP recombination. These vectors were transfected into permanent cell lines COS7, MDCK-iowa, T84 and CaCO2, in order to assess the effects of the genomic context elements upon EGFP. The proximal CFTR 5’ region in the p1kbcfproEGFP vector drove expression of the EGFP transgene at low levels in every cell line analysed. This is in agreement with previous reports that show basal levels of CFTR expression driven by this proximal ‘housekeeping’ region. The additional upstream region in the _PAC65bcfproEGFP vector did not appear to modulate expression in any of the cell lines analysed. A comparison of the twin vectors _PACRC1iresEGFP and _PACRC2iresEGFP, which differ only in the absence or presence of CFTR intron 1 respectively, showed similar levels of expression in the COS7 and MDCK-iowa cell lines. Thus, the intron 1 element does not seem to alter expression in these non-gut cell lines; this is consistent with reports that show regulation of CFTR expression in response to the intron element to be specific to cells of the gut epithelium. A comparison of pEGFP-N and _PACRC2cmvEGFP revealed that large PAC vectors show an intrinsic reduction in expression in comparison to their small plasmid counterparts. Further experiments showed that this was not an effect of vector copy number, and that the effect could not act in trans upon a co-transfected molecule. These studies also revealed an unexpected interaction: diluting a reporter plasmid with an anonymous plasmid may actually increase its transfection efficiency. An ex vivo primary air interface sheep tracheal culture was utilised as a more realistic model. Cultures were transfected with several of the genomic context vectors. While PAC vectors had shown a dramatic reduction in expression relative to their small plasmid counterparts in the in vitro studies, only a small reduction was seen to the ex vivo cultures, thus PAC vectors, such as GCVs, may provide a promising approach for gene therapy studies.

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Waller, Michael David. "The behaviour of the cystic fibrosis respiratory epithelium and its response to multidose CFTR gene therapy." Thesis, Imperial College London, 2016. http://hdl.handle.net/10044/1/41881.

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Cystic fibrosis (CF) is a clinical syndrome resulting from inherited mutations to the cystic fibrosis transmembrane conductance regulator (CFTR) protein, whose absent or reduced function results in abnormal epithelial ion transport and an abnormal transepithelial potential difference (PD), leading to downstream epithelial dysfunction and a pathognomonic clinical phenotype. Most treatments to date manage the disease sequelae of airway mucus and infection, but correction of the underlying defect for all patients is the ultimate aim. Gene therapy offers the potential as a universal treatment to ameliorate CFTR function, leading to clinical benefit. This thesis will centre on the recently completed Multidose Trial (MDT) - the repeated application of non-viral CFTR gene therapy in patients with cystic fibrosis. The trial was undertaken during the entirely of this PhD, and recruited 136 patients (aged ≥12) with CF, and randomised to receive 12, monthly nebulised doses of a cationic-lipid (pGM169/GL67A), or placebo. The hypothesis of the study was that repeated administration of non-viral gene therapy would produce vector-specific epithelial CFTR, leading to demonstrable improvements in lung function and measureable de novo chloride ion transport in the upper (nasal) and lower (bronchial) airway. The study reports stability in lung function with gene therapy (n=62) at 48 weeks compared to a decline in the placebo group (n=54), concluding a significant treatment effect of a relative improvement of 3.7% (p=0.046) in percent-predicted forced expiratory volume in 1 second (FEV1) at follow-up; other clinical parameters further support a treatment benefit, however a reduction in the frequency of pulmonary exacerbations was not detected. A post hoc analysis identified a significant treatment benefit of 6.4% in patients with more severe airways disease (ppFEV1 50-70%), however failed to detect a treatment effect in patients with less severe disease (ppFEV1 70-90%). Individual patients demonstrated de novo chloride transport in the nasal and lower airway, as measured by epithelial potential difference, with a significant difference being measured in the lower airway in response to gene therapy (-4.4 mV, p=0.03); no difference was identified in the upper airway in response to active treatment. The thesis will next explore the relationships of epithelial PD between the nasal and lower airway with measurements of lung function at baseline, and in response to treatment with gene therapy. At baseline, trends between sodium transport in the nasal epithelium and FEV1 and a lung clearance index (LCI) (p=0.01) were identified; no relationship was identified between chloride indices, or with any measurement from the lower airway. No relationship between the electrophysiology of the upper and lower airway epithelium was detected. The author will present a novel method used to maintain blinding whilst performing nasal PD (NPD) measurements during the MDT, using a 2-operator technique. This technique is reported as not inferior to the standard NPD method and supports its validity of its use for future studies, but provides caution that this method may take longer to perform and that more data may be excluded owing to poor NPD trace quality. The thesis concludes by describing two studies designed to further understand the performance and interpretation of PD measurements, and discussing the overall usefulness of airway PD as a clinical trial outcome. The first study investigates the amount of total chloride secretion (in healthy (non-CF) volunteers (n=18)) in the nasal epithelium by perfusing 'standard nasal' (with amiloride) and 'lower airway' (sans amiloride) solutions, reporting that more (approximately 50%) chloride is secreted in the presence of amiloride-containing solutions, and when the duration of perfusion was extended. The final study aimed to define the minimum period for performing sequential NPDs in the same (CF) patient, reporting that 4/8 (50%) CF patients basal PD had returned after 30 min, and that basal PD had returned by 60 min in all patients, concluding that repeated measurements could be made over a short timeframe for clinical and research studies.

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Ghosh, Arkasubhra. "Rational design of split gene vectors to expand the packaging capacity of adeno-associated viral vectors." Diss., Columbia, Mo. : University of Missouri-Columbia, 2007. http://hdl.handle.net/10355/4712.

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Thesis (Ph. D.)--University of Missouri-Columbia, 2007.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "December 2007" Includes bibliographical references.

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Buckley, Suzane Mary Kirstie. "An investigation into prenatal gene therapy for cystic fibrosis using intra-amniotic vector application to mouse models." Thesis, Imperial College London, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.408131.

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Harding-Smith, Rebekka. "Gene transfer vector development to treat lung disease." Thesis, University of Oxford, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.711729.

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Grzybowski, Brad. "A pseudotyped viral vector : hPIV3-HIV-1." Thesis, Georgia Institute of Technology, 2003. http://hdl.handle.net/1853/20932.

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Davies, Michael Gordon. "The development of end-point assays to assess the safety and efficacy of intra-pulmonary gene therapy in cystic fibrosis." Thesis, Imperial College London, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.520985.

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Dickey, David Derrick. "Strategies for improving adeno-associated viral infection of airway epithelial cells." Diss., University of Iowa, 2012. https://ir.uiowa.edu/etd/2858.

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Cystic fibrosis (CF) is a lethal autosomal recessive genetic disease caused by mutations in a single gene, the cystic fibrosis transmembrane conductance regulator (CFTR). CF affects multiple organ systems, but the major cause of morbidity and mortality is due to disease in the lungs. In theory, using gene therapy to deliver a correct copy of CFTR to the cells of the airway epithelium could result in a lifelong cure. Adeno-associated virus (AAV) is a single stranded DNA virus that is a promising candidate vector for gene therapy of multiple diseases, and numerous clinical trials are currently underway. Despite recent clinical successes, several challenges still impede wider application of AAV gene therapy to numerous diseases, including CF, as AAV-mediated gene transfer to the airways remains below the level needed for therapeutic efficacy for CF. We hypothesized that the low transduction efficiency of AAV in the airways could be overcome by using directed evolution of AAV in organotypic human and pig airway models, and in vivo in the lungs of pigs to select novel AAV capsid variants with improved infectious properties. We discovered a highly infectious, novel AAV that was a chimera of AAV2 and AAV5 with one point mutation (A581T) which we called AAV2.5T. We found that AAV2.5T mediated gene transfer significantly better than its parental serotypes, and corrected the chloride transport defect in CF human airway epithelial cultures. We determined that AAV2.5T developed increased binding to the apical surface of human airway epithelial cells, and that it has evolved to utilize specific 2,3N-linked sialic acid residues on the cell surface that mediate rapid internalization and subsequent infection. Thus, sialic acid serves as not just an attachment factor but is also required for AAV2.5T internalization, possibly representing an important rate-limiting step for other viruses that use sialic acids. Additionally, we utilized directed evolution in vivo in the lungs of pigs to select a novel AAV capsid that is identical to AAV2 except for five point mutations, which we called AAV2H22. We found that AAV2H22 mediated gene transfer to pig airway epithelial cultures significantly better than AAV2, and that it had evolved altered receptor binding. We also found that directed evolution in vitro in human and pig airway epithelial cultures results in the selection of distinct viruses for the two species, and that maintaining different selection stringencies results in the recovery of different AAV variants. Finally, we utilized Hoechst 33342, a DNA binding compound which was previously found to increase AAV transduction in cell lines, to increase AAV-mediated gene expression in primary human airway epithelia. We determined that the mechanism of this effect was due to activation of the CMV promoter. The findings from this research have significant implications for our understanding of AAV biology and for pulmonary gene therapy.

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Chen, Xuguang. "Cellular Uptake of DNA Nanoparticles and Regulation of Cell Surface Nucleolin." Case Western Reserve University School of Graduate Studies / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=case1244145515.

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26

Smith, David L. "Nocturnal hypoxaemia in cystic fibrosis." Thesis, University of Southampton, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.296267.

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27

Utley, Courtney, and Kristen L. McHenry. "Advances in Cystic Fibrosis." Digital Commons @ East Tennessee State University, 2016. https://dc.etsu.edu/etsu-works/2546.

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The purpose of this review was to identify the history of and advances in cystic fibrosis (CF). New treatment plans, medication developments, and a historical perspective of airway clearance therapy (ACT) will be presented. The importance of treatment compliance and time management in the care of cystic fibrosis patients will also be discussed. Furthermore, the development of cystic fibrosis clinics and the pivotal role they play in the treatment of the disease will be addressed. Lastly, a brief discussion concerning the need for and process of lung transplantation will be reported.

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Desgrandchamps, Daniel. "Antimicrobial therapy of Pseudomonas pulmonary exacerbations in cystic fibrosis /." [S.l : s.n.], 1986. http://www.ub.unibe.ch/content/bibliotheken_sammlungen/sondersammlungen/dissen_bestellformular/index_ger.html.

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29

Brouqueyre, Laurent. "Hydro-acoustic therapy : design, construction and testing." Thesis, Georgia Institute of Technology, 1999. http://hdl.handle.net/1853/18215.

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30

Adair, Jeanette. "Alternate channel therapy for the pancreatic disease of Cystic Fibrosis." Thesis, University of Newcastle Upon Tyne, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.251005.

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31

Smitley, Veronica Rose. "The Voice and Cystic Fibrosis: A Descriptive Case Study." Cleveland State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=csu1463089613.

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32

Hill, Alison Jane Margaret. "Mutation analysis and automated sequencing of the CFTR gene." Thesis, Queen's University Belfast, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.282187.

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33

Broakes-Carter, Fiona. "The developmental regulation of expression of the ovine CFTR gene." Thesis, University of Oxford, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.269950.

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34

Nuthall, Hugh. "Analysis of DNase I hypersensitive sites in the CFTR gene." Thesis, University of Oxford, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.298724.

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35

Hughes, David J. "Mutation characterisation and microsatellite haplotype analysis of the CFTR gene." Thesis, Queen's University Belfast, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.361278.

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36

Hadd, Wendi. "Genetics and the social body, testing for the cystic fibrosis gene." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape9/PQDD_0025/NQ47617.pdf.

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37

Hadd, Wendy. "Genetics and the (social) body testing for the cystic fibrosis gene /." [Montréal] : Université de Montréal, 1998. http://wwwlib.umi.com/dissertations/fullcit/NQ47617.

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Abstract:

Thèse (Ph. D.)--Université de Montréal, 1999.
"Thèse présentée à la Faculté des études supérieures en vue de l'obtention du grade de Philosophiae Doctor (Ph. D.) [sociologie]." Version électronique également disponible sur Internet.

38

GARCIA-FONKNECHTEN, NORIA. "Etude des transcrits du gene cftr. (cystic fibrosis transmembrane conductance regulator)." Paris 7, 1993. http://www.theses.fr/1993PA077156.

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Le gene cftr (cystic fibrosis transmembrane conductance regulator), constitue de 27 exons repartis sur 250 kb et dont l'alteration est responsable de la mucoviscidose, est essentiellement exprime dans les epitheliums respiratoires et digestifs. Cette expression est indetectable par northern-blot dans des cellules facilement accessibles comme les lymphocytes. Cependant, par la technique d'amplification de l'adnc par nested-pcr, nous avons mis en evidence et quantifie les transcrits cftr dans les lymphoblastes (transcription illegitime). Bien qu'ils soient en tres faible quantite, par cette methodologie nous obtenons du materiel en quantite suffisante pour l'analyse moleculaire de ces transcrits. C'est par cette analyse chez un patient homozygote pour la mutation majoritairement retrouvee dans cette maladie (f508), que nous avons prouve que ces transcrits etaient une source de materiel pathologique. Tres encourages par nos premiers resultats et afin d'etudier la totalite du transcrit cftr, nous avons developpe un protocole permettant d'amplifier l'adnc codant (4,2 kb) en six fragments chevauchants. Utilisant cette strategie nous exposons dans ce travail trois applications de cette methode sur l'arn obtenu a partir des lymphoblastes des patients atteints de mucoviscidose: (1) detection de mutations ponctuelles sur de grands fragments d'adnc, ce qui permet d'explorer plusieurs exons de facon simultanee; (2) mise en evidence de transcrits anormaux resultant d'une mutation d'epissage; (3) detection de transcrits anormaux, s'agissant le plus souvent de la perte d'exons, chez des malades ne presentant aucune mutation d'epissage. Ces transcrits seraient issus d'un epissage aberrant ou alternatif. Des transcrits depourvus des exons 9 ou 12 ont ete egalement detectes chez les sujets normaux. Enfin, pour mieux comprendre ce phenomene d'epissage alternatif ou aberrant nous avons etudie les transcrits depourvus de l'exon 9 dans le pancreas humain normal, l'un des sites d'expression du gene cftr. Cette etude nous a permis la mise en evidence de tels transcrits dans les tissus pancreatiques a differents ages du developpement embryonnaire, ainsi que chez l'adulte

39

Basalla, Joseph. "Identifying biosynthetic gene clusters whose products inhibit cystic fibrosis derived pathogens." Bowling Green State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1530795688583696.

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40

Moulin, Danielle S. "Regulation of expression of the CFTR gene." Thesis, University of Oxford, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.298347.

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41

Hull, Jeremy. "Mutation analysis and screening in the cystic fibrosis transmembrane conductance regulator gene." Thesis, University of Oxford, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.260734.

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42

Kimber, Wendy Louise. "Gene targeting in the mouse : introducing specific mutations associated with cystic fibrosis." Thesis, University of Edinburgh, 1995. http://hdl.handle.net/1842/20609.

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This thesis describes an attempt to create mouse models for CF bearing precise, CF-associated mutations as the only alteration to the murine Cftr gene, through use of the 'Hit and Run' gene targeting technique in mouse embryonal stem (ES) cells. This involves an initial targeting step in which an insertional targeting vector bearing the mutation to be introduced integrates through homologous recombination into Cftr. A negative selection then follows, enriching for those cells which have subsequently excised the vector and either reverted to the wild type genotype, or been converted to the desired genotype of the introduced mutation in the correct location as the only alteration to the gene. Vectors incorporating CF-associated mutations were introduced into ES cells and found to target Cftr at a higher frequency than had been previously reported for this region of the murine CF gene. Selection against the integrated vector in these targeted 'hit' clones was conducted with the unexpected result of 100% of resistant clones retaining the vector and the selection cassette. Close examination of these clones discovered no gross rearrangements, but a failure of methylation-sensitive restriction enzymes to cut within this region which could be induced or removed by growing the clone in negative or positive selection respectively. Further investigation of this phenomenon led to the conclusion that expression of the selection cassette was being modulated by methylation, and loss of the negative selection gene expression by this mechanism was occurring far more frequently than the desired loss through vector excision. The 'run' procedure was modified in view of this to reduce the high methylation-induced background, and clones were subsequently obtained which had survived the selection through vector excision.

43

Saferali, Aabida. "Genetic association and gene expression analysis of inflammatory genes in cystic fibrosis." Thesis, University of British Columbia, 2016. http://hdl.handle.net/2429/59277.

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Cystic fibrosis (CF) is characterized by a progressive decline in lung function due to airway obstruction, infection, and inflammation. CF patients are particularly susceptible to respiratory infection by a variety of pathogens, and the inflammatory response in CF is dysregulated and prolonged. This thesis identifies and characterizes BPI fold containing family A, member 1 (BPIFA1) and BPIFB1 as putative anti-inflammatory molecules in CF, and explores the CF inflammatory response to rhinovirus infection. BPIFA1 and BPIFB1 are proposed innate immune molecules expressed in the upper airways. We interrogated BPIFA1/BPIFB1 single-nucleotide polymorphisms in data from the North American genome-wide association study (GWAS) for lung disease severity in CF and discovered that the G allele of rs1078761 was associated with reduced lung function in CF patients. Microarray and qPCR gene expression analysis implicated rs1078761 G as being associated with reduced BPIFA1 and BPIFB1 gene expression, suggesting that decreased levels of these genes are detrimental in CF. Functional assays to characterize the role of BPIFA1 and BPIFB1 in CF indicated that these molecules do not have an anti-bacterial role against P. aeruginosa, but do have an immunomodulatory function in CF airway epithelial cells. To further investigate the mechanism of action of BPIFA1 and BPIFB1 during bacterial infection, gene expression was profiled using RNA-Seq in airway epithelial cells stimulated with P. aeruginosa and treated with recombinant BPIFA1 and BPIFB1. Viral infections are now recognized to play an important role in the short and long term health of CF patients. Rhinovirus is emerging as a lead viral pathogen although little is known about the inflammatory response triggered by rhinovirus in the CF lung. To investigate whether CF patients have a dysregulated response to rhinovirus infection, primary airway epithelial cells from CF and healthy control children were infected with rhinovirus and gene expression profiles were assessed by RNA-Seq. Although rhinovirus stimulation resulted significantly altered gene expression, the response to infection was not different in CF patients compared to healthy controls. However, CF cells had significantly higher rhinovirus levels than controls, indicating that CF patients may have a deficient antiviral response allowing for increased rhinovirus replication.
Medicine, Faculty of
Experimental Medicine, Division of
Graduate

44

Sheldon, Christopher David. "The effects of antibiotic therapy on Pseudomonas aeruginosa in adults with cystic fibrosis." Thesis, University of Southampton, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.387001.

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45

Gziut, Marta. "Investigating copper chelation with tobramycin as an anti-inflammatory therapy in cystic fibrosis." Thesis, University of Portsmouth, 2012. https://researchportal.port.ac.uk/portal/en/theses/investigating-copper-chelation-with-tobramycin-as-an-antiinflammatory-therapy-in-cystic-fibrosis(15d77cd6-686f-4e22-bcd5-83e9394c6229).html.

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Excessive neutrophilic inflammation of the airways in response to infection is characteristic for patients with CF. There is also an important but not fully understood role for platelets. Previous studies established increased copper levels in the circulation and in the sputum in CF. Inhaled tobramycin was suggested to have an anti-inflammatory effect beyond eradicating Pseudomonas aeruginosa. This study tested the hypothesis that tobramycin has anti-inflammatory and anti-oxidant efficacy due to its ability to bind copper into a copper-tobramycin complex. A copper-tobramycin complex was synthesised and the UV-VIS spectrum analysed. Neutrophil migration through a TNF-α-stimulated human lung microvascular endothelial cell layer towards thrombin-activated platelets was measured. The role of CFTRinh-172 on neutrophil transendothelial migration was assessed. Endothelial tobramycin uptake and CFTR expression were assessed using immunocytochemistry. Endothelial oxidative stress was measured using a fluorescent indicator. Neutrophils were stimulated to measure reactive oxygen species (ROS) production and neutrophil elastase (NE) activity, spectrophotometrically. Platelet and endothelium-derived NAP-2 and IL-8, respectively, contributed to neutrophil transendothelial migration. Copper-tobramycin was shown to be more effective than tobramycin in limiting migration of neutrophils. Both, tobramycin and copper-tobramycin accumulated in endothelial cells via a heparan sulphate-dependent mechanism, decreased intracellular ROS and increased endothelial surface CFTR expression. CFTRinh-172 failed to create an inflammatory profile in endothelium. Copper-tobramycin decreased extracellular superoxide released by activated neutrophils, and displaced NE from sites of encryption, making it more susceptible to inhibition by α1-antitrypsin. The antibiotic tobramycin was demonstrated to be a multi-potent drug with additional anti-inflammatory and anti-oxidant properties. These effects, desirable in CF treatment, are due to copper binding.

46

Smith, Emily M. "The Three-Dimensional Structure of the Cystic Fibrosis Locus: A Dissertation." eScholarship@UMMS, 2011. http://escholarship.umassmed.edu/gsbs_diss/744.

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The three dimensional structure of the human genome is known to play a critical role in gene function and expression. I used chromosome conformation capture (3C) and 3C-carbon copy (5C) techniques to investigate the three-dimensional structure of the cystic fibrosis transmembrane conductance regulator (CFTR) locus. This is an important disease gene that, when mutated, causes cystic fibrosis. 3C experiments identified four distinct looping elements that contact the CFTR gene promoter only in CFTR-expressing cells. Using 5C, I expanded the region of study to a 2.8 Mb region surrounding the CFTR gene. The 5C study shows 7 clear topologically associating domains (TADs) present at the locus, identical in all five cell lines tested, regardless of gene expression status. CFTR and all its known regulatory elements are contained within one TAD, suggesting TADs play a role in constraining promoters to a local search space. The four looping elements identified in the 3C experiment and confirmed in the 5C experiment were then tested for enhancer activity using a luciferase assay, which showed that elements III and IV could act as enhancers. These elements were tested against a library of human transcription factors in a yeast one-hybrid assay to identify potential binding proteins. Element III gave two strong candidates, TCF4 and LEF1. A literature search supported these transcription factors as playing a role in CFTR gene expression. Overall, this work represents a model locus that can be used to test important questions regarding the role of three dimensional looping on gene expression.

47

Smith, Emily M. "The Three-Dimensional Structure of the Cystic Fibrosis Locus: A Dissertation." eScholarship@UMMS, 2014. https://escholarship.umassmed.edu/gsbs_diss/744.

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Abstract:

The three dimensional structure of the human genome is known to play a critical role in gene function and expression. I used chromosome conformation capture (3C) and 3C-carbon copy (5C) techniques to investigate the three-dimensional structure of the cystic fibrosis transmembrane conductance regulator (CFTR) locus. This is an important disease gene that, when mutated, causes cystic fibrosis. 3C experiments identified four distinct looping elements that contact the CFTR gene promoter only in CFTR-expressing cells. Using 5C, I expanded the region of study to a 2.8 Mb region surrounding the CFTR gene. The 5C study shows 7 clear topologically associating domains (TADs) present at the locus, identical in all five cell lines tested, regardless of gene expression status. CFTR and all its known regulatory elements are contained within one TAD, suggesting TADs play a role in constraining promoters to a local search space. The four looping elements identified in the 3C experiment and confirmed in the 5C experiment were then tested for enhancer activity using a luciferase assay, which showed that elements III and IV could act as enhancers. These elements were tested against a library of human transcription factors in a yeast one-hybrid assay to identify potential binding proteins. Element III gave two strong candidates, TCF4 and LEF1. A literature search supported these transcription factors as playing a role in CFTR gene expression. Overall, this work represents a model locus that can be used to test important questions regarding the role of three dimensional looping on gene expression.

48

Terrell, Andrew S. "The effect of hydro-acoustic therapy on sputum production in patients with cystic fibrosis." Thesis, Georgia Institute of Technology, 2000. http://hdl.handle.net/1853/17076.

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49

Alteirac, Laurent N. "Clinical trial of hydro-acoustic therapy and conception of a second-generation hydro-acoustic chamber." Thesis, Georgia Institute of Technology, 2001. http://hdl.handle.net/1853/19659.

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50

Rowntree, Rebecca Kate. "Regulation of expression of the human cystic fibrosis transmembrane conductance regulator (CFTR) gene." Thesis, University of Oxford, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.393265.

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Dissertations / Theses on the topic 'Cystic fibrosis Gene therapy'

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