Дисертації з теми "Cystic fibrosis; gene therapy; lentivirus"
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1
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.
Повний текст джерелаАнотація:
"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.
2
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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3
Davies, Gwyneth. "Outcome measures for cystic fibrosis gene therapy clinical trials." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/28414.
Повний текст джерелаАнотація:
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.
4
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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5
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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6
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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7
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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8
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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9
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.
Повний текст джерелаАнотація:
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.
10
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.
Повний текст джерелаАнотація:
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.
11
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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12
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.
Повний текст джерелаАнотація:
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.
13
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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14
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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15
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.
Повний текст джерелаАнотація:
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.
16
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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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.
Повний текст джерелаАнотація:
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.
18
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.
Повний текст джерелаАнотація:
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.
19
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.
Повний текст джерелаАнотація:
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.
20
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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21
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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22
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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23
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.
Повний текст джерелаАнотація:
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.
24
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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25
Kremer, Karlea Lee. "Cystic Fibrosis gene therapy: methods for the optimisation of CFTR gene delivery." Thesis, 2010. http://hdl.handle.net/2440/63153.
Повний текст джерелаАнотація:
Gene therapy potentially holds the key for the treatment and cure of many genetic diseases, including cystic fibrosis. A number of delivery methods have been developed for the integration of a functional gene into the host genome, one of which is the use of a HIV-1 derived lentivirus, as is used in this thesis. However, a large number of issues need to be addressed before an effective gene therapy protocol can be developed, and some of these are described further in this thesis.
One such issue is that the response initiated by cells to the gene transfer vector need to be addressed, as organelles such as the proteasome and lysosome that break down foreign peptides and proteins may be involved in the degradation of our gene transfer vector, ultimately limiting the amount of gene transfer vector that is able to successfully integrate into the genome. Therefore, the potential use of proteasome and lysosome inhibitors for facilitating higher levels of gene transduction in vivo was investigated.
As this project uses a HIV-1 derived lentivirus for gene transfer, the use of an inhibitor of the IN1/PML innate antiretroviral response (Leptomycin B) was also assessed, again with the aim of increasing the efficiency, and hence level, of gene transfer obtained.
Using a robust animal model of disease is essential for testing lentivirus constructs containing the therapeutic gene and analysing phenotypic changes in disease. A mouse model of cystic fibrosis without gastrointestinal disease was bred to obtain a robust colony of mice that efficiently produce affected mice (CFTR knockout).
Visual analysis of therapeutic gene transfer in cystic fibrosis is often difficult due to the lack of antibodies available. Short DNA molecules that adopt a specific 3-D shape known as aptamers hold much potential as agents that can be developed to bind to the CFTR gene product. These can then be labelled and used in the same way as antibodies to probe tissues excised from animals treated with the therapeutic CFTR gene.
Essential to gene therapy is the development of methods for the consistent determination of lentivirus titre. As the production of lentivirus becomes more sophisticated with the use of multiple transgenes in a single virus preparation, the need for multiple assays to determine the titres of each individual virus component are required. Real time PCR assays were developed for each individual transgene for titre determination. A real time PCR assay for use with CHOK-1 cells was also developed for
comparison of real time PCR titre of a LacZ virus to the titre obtained using the traditional LacZ titre achieved via a staining assay in CHOK-1 cells. The use of a standard real time PCR assay for the determination of titre for all viruses- containing any transgene is essential to allow comparison by titre.
Determination of the level of gene expression required to achieve a therapeutic outcome in a cystic fibrosis mouse model is an important factor to consider, as high level expression in all cells may not achieve the best outcomes, and low level, ciliated-cell specific expression may in fact achieve a superior result. A range of different strength and cell targeted promoters (EF1α, pgk and K18) were tested for their effect on phenotypic correction of the cystic fibrosis knockout mouse model.
Lastly, targeting therapeutics to the disease affected areas is essential in achieving the best patient outcomes. As the main target organ in cystic fibrosis are the conducting airways of the lungs, delivery of our gene transfer vectors to the lungs as an aerosol is a necessary step towards moving this gene therapy protocol to the clinic. Aerosolisation of the treatment protocol was investigated in the rat lung for evidence of whether this is a viable means of lentiviral mediated gene delivery to the airways.Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2010
26
Stocker, Alice. "Towards gene therapy for cystic fibrosis airway disease: development of single-dose lentiviral gene transfer for lifetime airway expression." Thesis, 2010. http://hdl.handle.net/2440/65308.
Повний текст джерелаАнотація:
Cystic Fibrosis (CF) is the most common, fatal autosomal recessive disorder affecting the Caucasian population with a frequency of 1 in 2500 live births and has a current median survival age of approximately 33 years. Characteristics of CF include abnormalities in sweat glands, malnutrition, pancreatic disease and infertility. It is however, severe and chronic lung disease that currently accounts for greater than 95% of morbidity and mortality in CF patients. The CF transmembrane conductance regulator gene was discovered in 1989 and in vitro correction of the defect soon followed, providing the basis for gene therapy as a potential cure for CF lung disease. To date, the lack of an efficient gene transfer vector system combined with the physical barriers of the airway epithelium limit the successful application of CF gene therapy. The work described in this thesis utilised a unique gene therapy approach developed by the CF Gene Therapy Research Group, which involved airway pre-treatment followed by gene delivery. Pre-treatment was with the natural detergent lysophosphatidylcholine (LPC), followed by a single-dose of a HIV-1 based lentivirus (LV) vector in vivo. Previously studies found significant gene expression within airway tissues, but areas of cell damage were also sometimes evident. Initial work included examining the relationship between gene transfer, LPC dose and timing parameters, and airway epithelial damage. This study found that 0.3% LPC
followed 60 minutes later with the LV produced significant gene expression within the airway, with only mild airway epithelial disturbance observed. The longevity of LV-mediated gene expression was then evaluated in the nasal airway of C57Bl/6 mice using the LacZ marker gene. Treatment of mouse nasal airway epithelium with the LPC prior to instillation of a single dose of an LVLacZ vector produced significant LacZ gene expression in many mice for at least 18 months. The finding of gene expression in one mouse after 24 months indicated essentially lifetime gene expression had been achieved. We found that a single dose of LVLacZ produced immediate as well as lifetime mouse airway expression, confirming our hypothesis that use of an integrating vector extends transgene expression. Importantly, LVCFTR dosing achieved at least 12 months of CFTR expression, representing partial functional correction of the CFTR defect in CF knockout mice. These findings provide evidence that a single-dose Lentiviral gene transfer method may offer a novel in vivo therapeutic paradigm in the pursuit of a cure for CF airway disease.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2010
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Cmielewski, Patricia. "Longevity of airway gene therapy for cystic fibrosis : single and repeat lentiviral dosing." Thesis, 2013. http://hdl.handle.net/2440/82142.
Повний текст джерелаАнотація:
The promise of gene therapy as a treatment and/or cure for cystic fibrosis (CF) airway disease is yet to be fulfilled. Lentiviral (LV) vectors possess many of the properties that would satisfy the requirements for an effective clinical gene correction treatment; the capacity to hold the large CF transmembrane conductance regulator (CFTR) gene, pseudotyped envelopes that provide broad tropism for a range of cells and tissue types, the ability to transduce dividing and non-dividing cells, the potential for long-term gene expression from genomic integration, and the lack of pre-existing blocking antibodies for majority of the CF population. To determine the persistence of LV gene expression, the same mice were repeatedly assessed throughout their lifetimes. The utilization of the biological compound lysophosphatidylcholine (LPC) as a pre-treatment enhanced nasal airway gene expression of the HIV-1 based LV vector containing reporter genes, or the functional CFTR gene, in normal and CF mice in vivo. Nasal luciferase (Luc) gene expression from a single LPC/LV nasal dose was sustained for the life time of normal mice, possibly suggesting an involvement of stem/progenitor cells or long-lived terminally differentiated cells. In contrast, stable long-term Luc gene expression was detected in the lung airways without the requirement of LPC pre-treatment. The loss then re-emergence of lung luminescence in CF mice demonstrated that stem/progenitor cells were transduced. This was the first examination of persistence of LV reporter gene and functional gene expression, in individual CF mice over their lifetimes. CF mice treated with LPC/LV-CFTR demonstrated a significant partial functional correction of the nasal CFTR electrophysiological defect that was sustained for up to 1 year. Importantly, this significantly increased survival, close to that observed in normal mice. Since the level of functional expression diminished over time in CF mice the ability to re-dose and evade blocking host immune responses was addressed. Multiple doses of a LV vector over a short time frame were feasible but did not significantly increase expression compared to a single dose. Circulating antibodies to both the vector envelope and the transgene protein were detected after repeat dosing conducting over a longer time frame. The timing of additional LV vector doses may be crucial for effective boosting of waning gene expression. The addition of a transient immunosuppressive treatment did not significantly enhance the level of gene expression produced by a single dose, but did reduce circulating antibodies to both the delivered foreign transgene and to the pseudotyped envelope protein. The demonstration of longevity of gene expression, the functional correction of the CFTR defect, the substantial increase in CF animal survival, the ability to re-dose and the use of immune-suppression to reduce antibody production provides strong and specific support for the continued investigation of LV CFTR gene transfer towards a clinical gene therapy treatment for CF airway disease.Thesis (Ph.D.) -- -University of Adelaide, School of Paediatrics & Reproductive Health, 2013
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Lee, Benjamin Haeyul. "Gene delivery to human sweat glands: A model for cystic fibrosis gene therapy." 2006. http://link.library.utoronto.ca/eir/EIRdetail.cfm?Resources__ID=442229&T=F.
Повний текст джерела
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Huang, Louise NY. "In vitro and in vivo gene transfer to respiratory epithelium in experimental models of fetal gene therapy for cystic fibrosis." Thesis, 2005. http://hdl.handle.net/2429/16533.
Повний текст джерелаАнотація:
Fetal gene therapy is a novel technique for correction of monogenic disorders, such as cystic fibrosis. The aims of this study were to: i) develop small animal models for fetal gene transfer to respiratory epithelium; ii) to determine the ideal gestational timing of transfection in our models; iii) evaluate in vivo gene transfer techniques which are directly applicable to human fetal therapy; and iv) assess the durability of in vivo fetal gene transfer postnatally. Vesicular Stomatitis Virus-G (VSV-G) pseudotyped lentiviral vector was used, which contains Green Fluorescence Protein (GFP), as the reporter gene. Fetal tracheas from time-mated New Zealand White pregnant rabbits were harvested on gestational day 24, 25 and 26 (term=31d) and put in organ culture medium, then transfected with 1 x 10⁶ viral particles. Following in vitro transfection, fetal tracheas began to express marker gene as early as one day after infection, with peak expression noted by day 7 post transfection. Results were confirmed by Polymerase Chain Reaction (PCR) and Immunohistochemistry (IHC). Based on the observation that 4 days of in vitro culture was necessary to achieve substantial marker gene expression, we evaluated two in vivo injection techniques on or before day 26 of fetal gestation: i) fetal tracheal injection (TI) following hysterotomy and partial fetal delivery, and ii) amniotic injection (AI) without hysterotomy. After injection of 1 x 10⁶ virus particles, fetuses and their control littermates were delivered by caesarean section on gestational day 30. Fetal tissues (trachea, lung, gut, liver, kidney gonad) were harvested and marker gene expression was confirmed by fluorescent microscopy, PCR and IHC. By PCR, there was evidence of extensive transduction of fetal tissues by AI (trachea, lung, gut, liver, kidney, skin), yet selective transduction of trachea and lung by TI. IHC localized airway expression of GFP to tracheal surface epithelium and pulmonary alveolar cells. Control fetuses expressed marker gene following TI, presumably through blood borne transmission via placental vascular connections, while comparatively few control fetuses were positive for marker gene after AI. One doe was found to have GFP DNA in lung after TI, suggesting that maternal infection is possible with this model. Marker gene expression was also observed in mid-gestational fetal CD1 mice following AI. Transfected fetuses were survived and sacrificed at various post-natal time points to test the durability of gene expression by Quantitative real-time PCR. The marker gene was detectable as late as 21 days after birth; however there was evidence that although transgene was detectable by PCR in trachea 3 weeks after birth, there was no corresponding GFP mRNA, suggesting that transgene expression may be "switched off. Lentiviral vector mediated gene transfer using direct amniotic injection techniques exhibited transduction of respiratory epithelium in fetal rabbits and mice in vivo. This experimental system should prove useful for future fetal gene therapy studies.Surgery, Department of
Medicine, Faculty of
Graduate