Abstract 1369 Neonatal Disease Oriented Research: Steroids & Oxygen: Perinatal Effects Poster Symposium, Sunday, 5/2

Gene therapy is a potentially useful strategy to alter lung function. In some circumstances, targeting circumscribed areas of the lung may be useful. To this effect, we attempted transpulmonary delivery of a plasmid containing the full length rat HO-1 cDNA in a vector which contains expressed in a tetracycline regulatable, bidirectional promoter (pBI-L, Clontech). This vector allowed for maximal expression of HO-1 and luciferase when co-transfected with the Tet-Off plasmid or minimal expression of HO-1 and luciferase when co-transfected with the Tet-On plasmid, in the absence of doxycycline. Neonatal C57B1/6 mice aged 5 days were injected with a mixture of 12.5µg each of pBI-L/HO-1 and Tet-Off in 10 µl Lipofectin into the right lung. In other experiments, neonatal mice were similarly injected using pBI-L/HO-1 and Tet-On. Some animals were injected intraperitoneally twice daily with 2 mg/kg doxycycline to evaluate the ability to regulate gene expression by Tet-Off and Tet-On in vivo. After intraperitoneal injection of luciferin, photon emission from the injected lung was non-invasively visualized at daily intervals and quantitated with an intensified CCD camera (Hamamatsu) to determine the duration of gene expression after a single injection. Pups injected with pBI-L/HO-1 and Tet-Off were exposed to either air or hyperoxia for 48 hours to document whether we could detect a physiologic effect of localized HO-1 injection in the lung. In these mice, gene expression was visualized, levels of HO-1 immunoreactive protein assessed and protein carbonyls measured as an index of oxidative injury in three lobes of the lung (upper, middle and lower). With administration of doxycycline, gene expression was decreased in the Tet-Off and increased in the Tet-On transfected lungs. With injection of the pBI/L-HO-1 + Tet-Off plasmids, HO-1 and luciferase gene expression was consistently elevated as compared to the uninjected lung over the course of 8 days. With 48 hours of hyperoxic exposure, luciferase gene expression was further increased. The HO-1 immunoreactive protein was highest in hyperoxia exposed animals and in particular in the middle lobe of the lung where the gene had been delivered. Furthermore, increased HO-1 expression was associated with the highest protein carbonyl content. This increased expression of HO-1 and increased protein oxidation was not observed in similarly exposed animals injected with a control plasmid, pGL-3/CMV containing the luciferase gene but not the HO-1 cDNA, indicating that the pro-oxidant effect observed was associated with HO-1 over-expression and not merely with transfection and expression of foreign genes. We conclude that localized injection of a small amount of DNA can result in significant, prolonged gene expression in the lung. In addition, gene expression can be regulated in vivo with doxycycline and this altered gene expression may be associated with circumscribed physiologic effects in the lung.