Enhanced Distribution of Adenovirus-Mediated Gene Transfer to Lung Parenchyma by Perfluorochemical Liquid

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Abstract
Although gene therapy holds great promise for the treatment of inherited and acquired diseases of the lung, a number of issues including efficient delivery and distribution of genes to pulmonary target cells must still be addressed. In this study we evaluated the use of perfluorochemical (PFC) liquid as a vehicle for delivery of recombinant adenovirus (AdCBlacZ) to lungs of juvenile rabbits. Virus was instilled into trachea of rabbits, and 4 days later the lungs were removed, cut into multiple pieces, and assayed for β-galactosidase (β-Gal) activity. Total lung expression of the β-Gal reporter gene was increased two- to three-fold by instillation of the virus (1011 particles/kg body weight) in saline (1.5 ml/kg) simultaneously with perflubron liquid (15 ml/kg) compared to virus + saline alone (control). Uniformity of β-Gal activity between lobes was significantly improved by the PFC liquid. In perflubron-treated lungs ~45% of the lung pieces had β-Gal-specific activity values within 50–150% of the mean specific activity for the total lung, compared to only ~15% of the pieces in control lungs. More of total lobar β-Gal activity was recovered in the distal lung tissue (~two-fold greater than controls, p < 0.05). Morphological assessment of X-Gal-stained, fresh-frozen lung sections showed increased levels and more complete staining of alveolar wall cells in the PFC group. These data indicate that the PFC liquid perflubron enhances distribution of virus-mediated gene expression to the lung parenchyma in healthy rabbits. PFC liquid may be a useful treatment vehicle for accessing distal spaces of the damaged or diseased lung. The clinical application of gene therapy faces several problems, one of which is the efficient distribution of genes to the desired target cells. We hypothesized that the particular properties of perfluorochemical liquids (PFC) make these compounds useful vehicles for distributing gene products to lung alveolar cells in distal airspaces. Young rabbits were instilled intratracheally with the β-galactosidase (β-Gal) reporter gene in an adenoviral vector (AdCBlacZ, 1011 particles/kg) using saline (1.5 ml/kg, control) or saline + perflubron (LiquiVent 15 ml/kg) to fill lungs to approximate functional residual capacity (FRC). Compared to saline alone, delivery of virus with perflubron increased total lung β-Gal activity (A420 units/mg protein) two- to three-fold and improved both interlobar and intralobar distribution of gene expression. By morphological assessment, use of perflubron also increased alveolar localization and uniformity of X-Gal staining in lung parenchyma. PFC liquid shows promise as a vehicle for efficient delivery of adenoviral vector to the distal lung.