Targeting transgene expression for cystic fibrosis gene therapy.

We have developed an expression cassette for cystic fibrosis (CF) gene therapy using control elements from the human cytokeratin 18 gene (KRT18, also known as K18). KRT18 is naturally expressed in a spatial pattern similar to that of CFTR, the gene mutated in CF. We delivered a KRT18-driven lacZ plasmid complexed with cationic liposomes intravenously to mice and examined expression in various tissues. We found expression in nasal and bronchial epithelium, airway submucosal glands, gall bladder, and kidneys. Expression was low in pancreas and gut, and absent from liver and alveolar lung. This is consistent with the expression pattern reported for a K18lacZ transgenic mouse. Following delivery of a cytomegalovirus (CMV) major immediate-early promoter/enhancer-driven lacZ plasmid, we found expression in bronchi, submucosal glands, alveolar cells, liver, and kidney. We did not detect expression in nose, pancreas, gall bladder, or gut. Using fluorescently labeled plasmid delivered by means of liposomes, we identified the liver, alveolar lung, and kidneys as the major plasmid deposition sites. Our data demonstrate that a KRT18-driven expression vector delivered systemically can target gene expression to CF-affected tissues, despite an uneven distribution of plasmid DNA. A KRT18-based vector may be a useful alternative to viral promoter-based vectors in clinical gene therapy trials to treat CF.

Comparison between intratracheal and intravenous administration of liposome-DNA complexes for cystic fibrosis lung gene therapy.

Intratracheal (i.t.) and intravenous (i.v.) delivery of DNA-vector formulations are two strategies to obtain gene transfer to the lung, it is still uncertain, however, which of these two modes of delivery will be more effective in the treatment of cystic fibrosis and other lung diseases. In this study, we attempted to optimize formulations of the cationic liposome DODAC:DOPE (dioleoyldimethylammonium-chloride: dioleoylphosphatidylethanolamine) complexed to plasmids encoding chloramphenicol acetyltransferase for i.t. and i.v. injection into CD-2 mice and compared the two methods. Our results showed that both methods conferred reporter gene expression in the lung that was significantly higher relative to injection of plasmid DNA alone. Expression using either mode of administration was maximal 24 h after injection and declined to around 10% of day 1 levels 2 weeks after injection. For i.v. delivery of DODAC. DOPE-DNA complexes multilamellar vesicles were more effective than large unilamellar vesicles in all organs investigated. Recombinant DNA could be detected in the distal lung region following either route of administration. However, i.t. administration predominantly led to DNA deposition in epithelial cells lining the bronchioles, e.g. in clara cells, whereas i.v. administration resulted in DNA deposition in the alveolar region of the lung including type II alveolar epithelial cells.

Active 22Na+ transport by the intact lung during early postnatal life.

The lung relies upon epithelial active transport of Na+ to aid in the clearance of fluid from its air spaces. Because it is unknown whether the rate of active Na+ transport by the distal lung epithelium varies during early postnatal age, we performed studies in young guinea pigs (7 and 30 days after birth). We used a single pass isolated perfused lung model in which a Krebs Ringer bicarbonate solution containing 22Na+, [14C]sucrose, and FITC-dextran was placed into the air spaces of the lungs, and apparent permeability-surface area (PS) products were calculated after determining the changes in lung weight and the concentrations of the isotopes in the vascular effluent. The PS product for 22Na+, but not [14C]sucrose, decreased significantly at both ages when amiloride was infused (final concentration of 10(-4) M). Amiloride also decreased the rate of fluid clearance, as assessed by changes in organ weight, at both ages. Although the absolute rate of amiloride-sensitive 22Na+ transport increased with age, morphometric measurement of the alveolar region demonstrated that the rate of amiloride-sensitive 22Na+ transport per unit alveolar surface area was similar. These data indicate that although the guinea pig lung undergoes significant growth shortly after birth, the rate of amiloride-sensitive active Na+ transport per unit surface area remains constant. Since a component of weight loss was insensitive to amiloride, these in vivo studies suggest that the amiloride-insensitive Na+ transport pathways previously identified in cultured lung epithelium exist in the intact lung.

Exogenous surfactant rapidly increases PaO2 in mature rabbits with lungs that contain large amounts of saline.

Neonatal respiratory distress syndrome (RDS) is characterized by a relative surfactant deficiency and air-space edema. We tested the hypothesis that exogenous surfactant would improve gas exchange in mature surfactant-replete lungs containing large amounts of saline. Healthy young rabbits weighing 550 to 1,000 g were anesthetized and tracheotomized, and they received assisted ventilation with a FlO2 = 1. Baseline PaO2 decreased when 20 ml/kg of warmed saline (n = 6) was instilled into the lung; average PaO2 was stable and remained less than 200 mm Hg during the next 180 min. When either 50 mg/kg of natural surfactant (n = 6) or lipid-extracted surfactant (LES) (n = 6) was included in the saline solution, the PaO2 was higher (p < 0.05) than in the saline-alone group. To evaluate the effect of delayed addition of the surfactant we performed six additional experiments; the PaO2 fell to 60 +/- 6.9 SEM mm Hg after saline instillation, but within 15 min of administering LES, the PaO2 increased to 246 +/- 44.1 mm Hg, and it rose to 469 +/- 29.6 mm Hg by the end of the experiment (t = 180 min). Similar ventilator settings maintained comparable PaCO2 values in untreated and surfactant-treated groups. Gravimetric lung water contents (ml/kg body weight or ml/total lung hydroxyproline content) were markedly increased, but similar, in all groups. These studies show that the increase in PaO2 after surfactant administration to mature lungs containing large amounts of saline is similar to the increase in PaO2 seen when surfactant is given to premature infants with RDS.(ABSTRACT TRUNCATED AT 250 WORDS)

Sodium channel but neither Na(+)-H+ nor Na-glucose symport inhibitors slow neonatal lung water clearance.

Normal clearance of alveolar liquid following birth requires active Na transport; however, the contribution of Na channels, Na-H antiports, and Na-glucose symports is unknown. We demonstrated that intraalveolar instillation of amiloride (n = 6) or the more specific Na channel blockers benzamil (n = 13) or phenamil (n = 12) before the first breath impaired lung water clearance relative to control newborns (n = 34). Benzamil and phenamil were more potent than amiloride (P less than 0.05). Neither the Na-H antiport inhibitor dimethyl amiloride (n = 7) nor the Na-glucose symport inhibitor phloridzin (n = 7) impaired lung water clearance. Ion substitution experiments with fetal rat type II alveolar epithelia demonstrated that more than 95% of their resting or terbutaline-stimulated short circuit current (Isc) depended upon Na bathing their apical membrane. Isc was decreased by amiloride (IC50 of amiloride-sensitive Isc = 0.3 x 10(-6) M) and benzamil (IC50 of benzamil-sensitive Isc = 0.3 x 10(-7) M) but was unaffected by dimethyl amiloride (10(-4) M). We conclude that in vivo postnatal clearance of fetal lung liquid can be impaired by Na channel blockers and is unaffected by blockers of Na-H antiports and Na-glucose symports. Na transport in fetal type II cells has high affinity for amiloride, and these cells likely contribute to normal neonatal lung liquid clearance.

Effect of pentoxifylline on hemodynamics, alveolar fluid reabsorption, and pulmonary edema in a model of acute lung injury.

We investigated the effect of pentoxifylline (PTX) on the development of pulmonary edema in a model of adult respiratory distress syndrome in rabbits. Lung injury was induced by repeated saline lavages in adult rabbits weighing 2.5 to 3.5 kg. Rabbits pretreated with PTX (20 mg/kg bolus followed by 20 mg/kg/h infusion) developed significantly lower amounts of lung edema 4 h after saline lavage (extravascular lung water to dry weight ratio [W/D], 6.9 +/- 0.6 SD versus 8.9 +/- 0.5 in control animals). PTX produced a 25% increase in cardiac output, but there were no differences between treated and untreated groups in calculated pulmonary vascular resistance or microvascular pressure. To determine whether PTX could have lowered pulmonary venous resistance and thus lowered effective microvascular pressure for fluid filtration, we directly measured pulmonary artery and left atrial pressures, and measured by micropuncture the pressure in 20 to 40 microns subpleural venules in four open-chested rabbits 3 to 4 h after lavage. Venous resistance was low (venous pressure drop 0.9 +/- 0.1 mm Hg) and was unchanged by PTX infusion. To determine if PTX decreased lung water by accelerating active alveolar fluid reabsorption, a single 60-ml aliquot of saline was instilled into the lungs of normal rabbits treated with saline or PTX. Both groups had a similar decrease in lung water content 1 and 4 h later. Our data indicate that PTX reduces edema formation in rabbits after saline lavage, not by lowering microvascular pressures for fluid filtration or by acceleration alveolar fluid reabsorption, but possibly by its anti-inflammatory effect on neutrophil function.

Amiloride impairs lung water clearance in newborn guinea pigs.

To determine whether epithelial ion transport is physiologically important for lung water clearance after birth, the sodium transport inhibitor amiloride or its vehicle saline was given intratracheally to newborn full-term guinea pigs before the first breath. Guinea pigs given saline intratracheally breathed normally and had arterial O2 saturations (SaO2) greater than 94%. In contrast, guinea pigs that had an estimated 10(-4) M intra-alveolar concentration of amiloride had chest wall retractions and 88 +/- 3.6% (SD) SaO2 (P less than 0.01). Extravascular lung water (EVLW) per gram of dry lung weight 4 h after birth was significantly greater in newborns that received amiloride (8.3 +/- 1.1, n = 5) than in those that received saline (5.6 +/- 0.9, n = 7, P less than 0.01). The degree of perivascular fluid cuffing at 25 cmH2O inflation was quantitatively similar in amiloride- and saline-treated animals. The effect of amiloride was dose dependent. Intratracheal amiloride did not affect EVLW in 9-day-old guinea pigs. This study demonstrates that intratracheal amiloride before the first breath results in respiratory distress, hypoxemia, and an abnormally high EVLW. Epithelial sodium transport contributes normal lung liquid clearance after birth.