685. Single-Dose Lentivirus Gene Transfer In Vivo Maintains Airway Gene Expression for over Six Months

Top of pageAbstract Introduction: The use of gene therapy as an effective treatment for Cystic Fibrosis (CF) airway disease has been limited by poor longevity of gene expression and inefficient gene transfer. Using a lysophosphatidylcholine pre-treatment method our group has documented significant increases in the efficiency of gene expression using the LacZ reporter gene (Limberis et al., 2002), but longevity of expression was not studied past 3 months. In this study we have confirmed and extended those findings and show that single dose exposure of murine nasal airway to a lentiviral (LV) vector carrying the LacZ gene (LVLacZ) is maintained for at least 6 months. Methods: A single 4|[mu]|l dose of LPC (0.3% w/v) was instilled into the right nostril of anaesthetised C57Bl/6 mice. One hour later LVLacZ vector (20 |[mu]|l bolus) was instilled into the same nostril. At four time-points post delivery (1 week, 1, 3 and 6 months, n=7 |[ndash]| 12 per group) mice were sacrificed and processed to reveal LacZ gene expression in ciliated respiratory and transitional airway epithelium via standard sectioning and x-gal procedures. Results: Across all assessment time points LVLacZ instillation gene expression was statistically equal to the initial (1 week) level of expression, though there was a trend towards a small reduction of expression between 1 week and 1 month. At both the 3 and 6 month time points several mice in these groups showed absence of gene expression (3 out of 12 mice and 2 out of 12 mice respectively), but this did not occur in the 1 week or 1 month groups where all mice displayed notable levels of gene expression. Conclusions: Epithelial cell turnover in murine airway has been shown by others to be approximately 3 months. Thus, the persistence of gene expression at levels equal to those seen at 1 and 3 months, for more than 6 months supports the notion that progenitor cells have been transduced by our gene delivery protocols. We speculate that the complete absence of gene expression in a small subset of mice in the 3 and 6 month groups, but not in the 1 week or 1 month groups, may represent failures in the delivery of LVLacZ to airway progenitor cells, resulting in absence of gene expression at time points longer that the renewal time for this epithelium. Experiments to determine longevity of gene expression at longer time points are in progress.