Towards gene therapy for cystic fibrosis airway disease: development of single-dose lentiviral gene transfer for lifetime airway expression.
Date
2010
Authors
Stocker, Alice
Editors
Advisors
Parsons, David Webb
Anson, Donald Stewart
Anson, Donald Stewart
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Type:
Thesis
Citation
Statement of Responsibility
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Abstract
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.
School/Discipline
School of Molecular and Biomedical Science
Dissertation Note
Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2010
Provenance
Copyright material removed from digital thesis. See print copy in University of Adelaide Library for full text.