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|Title:||Development of lentiviral airway gene therapy aerosol delivery techniques for cystic fibrosis|
|School/Discipline:||Adelaide Medical School|
|Abstract:||Lentiviral (LV) vectors show promise as a gene therapy vector for cystic fibrosis (CF). The cystic fibrosis airway gene therapy group (CFARG) based in Adelaide, Australia have developed a HIV-1 based LV vector that demonstrated expression of the corrective CFTR gene up to 12 months in the airways of CF mice. Using their two-step airway conditioning and bolus gene vector delivery technique, the CFARG have shown effective transduction in the airways of animal models such as sheep and marmosets. As this vector approaches clinical realisation there is a need to translate the bolus delivery regimen of this vector to an aerosol form. Aerosolisation would enable non-invasive and easily repeatable vector delivery, which could be used in future clinical trials. This thesis examines the efficiency of different delivery devices for aerosolising the LV vector. Cell culture studies showed that LV vector aerosolised using a newly developed ultrasonic surface acoustic wave (SAW) nebuliser produced significantly lower levels of gene expression than bolus delivery. This led to examination of an intra-tracheal sprayer, the MADgic™ atomisation device, which demonstrated promising results on delivering the vector as a spray, in cell culture studies. However, use of this device in human clinical studies is invasive. Therefore, the efficacy of delivering LV vector as an aerosol through other nebulisers was investigated. A baseline in vivo study was designed to aerosolise the LV vector into the lungs of mechanically ventilated mice using an Aeroneb®Pro vibrating mesh nebuliser with a flexiVent™ small animal ventilator. This was the first study to compare the levels of gene expression produced by a HIV-based LV vector delivered either as an aerosol or as a bolus dose into mouse lungs. Lower levels of gene expression were obtained in the trachea of aerosol-treated animals compared to bolus-treated animals. However, the effect of LV aerosol delivery could not be determined in other conducting airways or the lung parenchyma, due to low power of the study produced by a substantial outlier producing a far larger than expected variability. The reason for the lowered gene expression observed in the trachea of mice treated with LV vector delivered as an aerosol through the Aeroneb®Pro-flexiVent™ ventilator apparatus was not conclusive. Further experiments investigated the cause of the low levels of gene expression observed in the baseline in vivo study. Bench studies with dye solution revealed that the physical dose volume that reached the tip of the endotracheal (ET) tube following delivery through the ventilator circuit was only 2% of the initial dose volume, which likely explained the low levels of gene expression in trachea of aerosol-treated animals. The delivery parameters were therefore optimised to increase the aerosol output available at the end of the delivery circuit. Subsequent cell culture studies examined the gene expression produced by the LV vector aerosolised with parameters used in the baseline in vivo study. The results demonstrated lowered gene expression produced by LV vector released at the end of the ventilator circuit compared to bolus delivery. To address this problem, different protective diluents were tested to try to preserve LV viability following aerosol delivery. Of these, FreeStyle™ medium produced higher levels of gene expression than our standard diluent, mouse serum in saline. On further examination, the LV vector suspended in an optimal diluent combined with optimal aerosolisation parameters produced higher levels of gene expression compared to baseline in vivo delivery parameters. However, despite these improvements the levels of gene expression produced by LV aerosol delivery was still significantly lower than bolus delivery. Hence, these studies indicate that the Aeroneb®Pro nebuliser was not ideal for aerosolising the LV vector in its current formulation. Together the results presented in this thesis highlighted the problems associated with aerosolising a HIV-based LV vector using different nebulisers and through a small-animal ventilator circuit. Although the nebulisers investigated in this thesis were not efficient for aerosolising LV vector, an intra-tracheal sprayer suitable to deliver this vector for testing in larger animal models was identified. Future research should examine other newly introduced nebulisers to develop an optimal vector delivery protocol for use in CF airway gene therapy.|
|Dissertation Note:||Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2019|
|Provenance:||This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at: http://www.adelaide.edu.au/legals|
|Appears in Collections:||Research Theses|
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