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Title: Strategies to increase gene transfer to the lung
Author: Larsen, Mia Damm Brygger
ISNI:       0000 0004 2698 5186
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2011
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Although proof-of-principle for non-viral gene transfer to the airways of cystic fibrosis patients has been established the efficiency is comparatively low. Two strategies related to further improving gene transfer were studied in this PhD. First, bacteria-mediated gene transfer (bactofection) and second, the ability of tight junction openers to enhance liposome-mediated gene transfer to murine lung in vivo were assessed. A genetically engineered E. coli strain was used to assess bactofection. Successful in vitro bactofection was demonstrated. In vivo data indicated that E. coli-mediated lung bactofection does not lead to easily detectable eukaryotic gene expression, and is less efficient than well established GL67-mediated gene transfer. A narrow toxicity/efficacy window was identified and, furthermore, reporter gene expression in the bacterial host complicated interpretation of the data. The ability of ethyleneglycol-bis-(β-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), lysophosphatidylcholine (LPC), sodium caprate (C10) and sodium laurate (C12) to enhance GL67-mediated gene transfer in vivo by modulating tight junctional permeability was assessed. EGTA had no effect. Pre-treatment with C10, C12 and LPC significantly enhanced gene transfer to murine lung and nose after careful optimisation of drug doses and delivery. C10 proved most efficient and potential difference measurements indicated that C10 altered tight junction permeability in the murine nasal epithelium. However, pre-treatment with C10 did not allow correction of the ion transport defect in CF mice or enhance CFTR mRNA expressions levels. A higher concentration of C10 was also assessed in the murine nose; tissue damage was observed, indicating a narrow efficacy/toxicity window. Finally, in a human airway ex vivo model, C10 pre-treatment resulted in a significant decrease in transepithelial resistance indicating an opening of tight junctions, but no enhancement of GL67-mediated gene transfer was observed. In conclusion, bactofection does not provide any advantages over lipid-mediated gene transfer. Lipid-mediated gene transfer can be enhanced through administration of tight junction openers and, therefore, may warrant further assessment in large animal models.
Supervisor: Alton, Eric ; Griesenbach, Uta Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral