Restoring ciliary function to differentiated primary ciliary dyskinesia cells with a lentiviral vector.

Primary ciliary dyskinesia (PCD) is a genetically heterogenous autosomal recessive disease in which mutations disrupt ciliary function, leading to impaired mucociliary clearance and life-long lung disease. Mouse tracheal cells with a targeted deletion in the axonemal dynein intermediate chain 1 (Dnaic1) gene differentiate normally in culture but lack ciliary activity. Gene transfer to undifferentiated cultures of mouse Dnaic1(-/-) cells with a lentiviral vector pseudotyped with avian influenza hemagglutinin restored Dnaic1 expression and ciliary activity. Importantly, apical treatment of well-differentiated cultures of mouse Dnaic1(-/-) cells with lentiviral vector also restored ciliary activity, demonstrating successful gene transfer from the apical surface. Treatment of Dnaic1(flox/flox) mice expressing an estrogen-responsive Cre recombinase with different doses of tamoxifen indicated that restoration of ∼20% of ciliary activity may be sufficient to prevent the development of rhinosinusitis. However, although administration of a ß-galactosidase-expressing vector into control mice demonstrated efficient gene transfer to the nasal epithelium, treatment of Dnaic1(-/-) mice resulted in a low level of gene transfer, demonstrating that the severe rhinitis present in these animals impedes gene transfer. The results demonstrate that gene replacement therapy may be a viable treatment option for PCD, but further improvements in the efficiency of gene transfer are necessary.

Transgenic hCFTR expression fails to correct ß-ENaC mouse lung disease.

The relationships between airway epithelial Cl(-) secretion-Na(+) absorption balance, airway surface liquid (ASL) homeostasis, and lung disease were investigated in selected transgenic mice. 1) To determine if transgenic overexpression of wild-type (WT) human CFTR (hCFTR) accelerated Cl(-) secretion and regulated Na(+) absorption in murine airways, we utilized a Clara cell secretory protein (CCSP)-specific promoter to generate mice expressing airway-specific hCFTR. Ussing chamber studies revealed significantly (∼2.5-fold) elevated basal Cl(-) secretory currents in CCSP-hCFTR transgenic mouse airways. Endogenous murine airway Na(+) absorption was not regulated by hCFTR, and these mice exhibited no lung disease. 2) We tested whether hCFTR, transgenically expressed on a transgenic mouse background overexpressing the ß-subunit of the epithelial Na(+) channel (ß-ENaC), restored ion transport balance and ASL volume homeostasis and ameliorated lung disease. Both transgenes were active in CCSP-hCFTR/ß-ENaC transgenic mouse airways, which exhibited an elevated basal Cl(-) secretion and Na(+) hyperabsorption. However, the airway disease characteristic of ß-ENaC mice persisted. Confocal studies of ASL volume homeostasis in cultured tracheal cells revealed ASL autoregulation to a height of ∼6 µm in WT and CCSP-hCFTR cultures, whereas ASL was reduced to <4 µm in ß-ENaC and CCSP-hCFTR/ß-ENaC cultures. We conclude that 1) hCFTR overexpression increases basal Cl(-) secretion but does not regulate Na(+) transport in WT mice and 2) transgenic hCFTR produces increased Cl(-) secretion, but not regulation of Na(+) channels, in ß-ENaC mouse airways and does not ameliorate ß-ENaC mouse lung disease.

Ion transport across CF and normal murine olfactory and ciliated epithelium.

The nasal epithelium of the cystic fibrosis (CF) mouse has been used extensively in CF research because it exhibits ion transport defects similar to those of human CF airways. This tissue is composed of approximately 50% olfactory (OE) and approximately 50% ciliated epithelium (CE), and on the basis of previous observations, we hypothesized that a significant fraction of the bioelectric signals from murine nasal tissue may arise from OE rather than CE, while CE is the target tissue for CF gene therapy. We compared the bioelectric properties of isolated OE from the nasal cavity and CE from the nasopharynx in Ussing chamber studies. Hyperabsorption of Na(+) [amiloride response; CF vs. wild type (WT)] was approximately 7.5-fold greater in the OE compared with the CE. The forskolin response in native tissues did not reliably distinguish genotypes, likely due to a cyclic nucleotide-gated cation conductance in OE and a calcium-mediated Cl(-) conductance in CE. By potential difference assay, hyperabsorption of Na(+) (CF vs. WT) and the difference in response to apical 0 Cl(-) buffer (CF vs. WT) were approximately 2-fold greater in the nasal cavity compared with the nasopharynx. Our studies demonstrate that in the CF mouse, both the hyperabsorption of Na(+) and the Cl(-) transport defect are of larger magnitude in the OE than in the CE. Thus, while the murine CF nasal epithelium is a valuable model for CF studies, the bioelectrics are likely dominated by the signals from the OE, and assays of the nasopharynx may be more specific for studying the ciliated epithelium.

Expression of CFTR from a ciliated cell-specific promoter is ineffective at correcting nasal potential difference in CF mice.

Successful gene therapy will require that the therapeutic gene be expressed at a sufficient level in the correct cell type(s). To improve the specificity of gene transfer for cystic fibrosis (CF) and other airway diseases, we have begun to develop cell-type specific promoters to target the expression of transgenes to specific airway cell types. Using a FOXJ1 promoter construct previously shown to direct transgene expression specifically to ciliated cells, we have generated transgenic mice expressing human cystic fibrosis transmembrane conductance regulator (CFTR) in the murine tracheal and nasal epithelia. RNA analysis demonstrated levels of CFTR expression is greater than or equal to the level of endogenous mouse CFTR. Immunoprecipitation and western blotting demonstrated the production of human CFTR protein, and immunochemistry confirmed that CFTR was expressed in the apical region of ciliated cells. The transgenic animals were bred to CFTR null mice (Cftr(tm1Unc)) to determine if expression of CFTR from the FOXJ1 promoter is capable of correcting the airway defects in Cl(-) secretion and Na(+) absorption that accompany CF. Isolated trachea from neonatal CF mice expressing the FOXJ1/CFTR transgene demonstrated a correction of forskolin-stimulated Cl(-) secretion. However, expression of human CFTR in ciliated cells of the nasal epithelia failed to significantly change the nasal bioelectrics of the CF mice.

Culture of murine nasal epithelia: model for cystic fibrosis.

The ion transport defects reported for human cystic fibrosis (CF) airways are reproduced in nasal epithelia of the CF mouse. Although this tissue has been studied in vivo using the nasal potential difference technique and as a native tissue mounted in the Ussing chamber, little information is available on cultured murine nasal epithelia. We have developed a polarized cell culture model of primary murine nasal epithelia in which the CF tissue exhibits not only a defect in cAMP-mediated Cl- secretion but also the Na+ hyperabsorption and upregulation of the Ca2+-activated Cl- conductance observed in human airways. Both the wild-type and CF cultures were constituted predominantly of undifferentiated cuboidal columnar cells, with most cultures exhibiting a small number of ciliated cells. Although no goblet cells were observed, RT-PCR demonstrated the expression of Muc5ac RNA after approximately 22 days in culture. The CF tissue exhibited an adherent layer of mucus similar to the mucus plaques reported in the distal airways of human CF patients. Furthermore, we found that treatment of CF preparations with a Na+ channel blocker for 7 days prevented formation of mucus adherent to epithelial surfaces. The cultured murine nasal epithelial preparation should be an excellent model tissue for gene transfer studies and pharmacological studies of Na+ channel blockers and mucolytic agents as well as for further characterization of CF ion transport defects. Culture of nasal epithelia from DeltaF508 mice will be particularly useful in testing drugs that allow DeltaF508 CFTR to traffic to the membrane.

Mucociliary transport determined by in vivo microdialysis in the airways of normal and CF mice.

We report a novel method to measure mucociliary transport (MCT) in both the upper and lower airways of normal and CF mice. The in vivo microdialysis technique involves placing a small quantity of dye on the airway surface and a microdialysis probe a defined distance from the site of dye deposition. The dye is transported toward the probe by ciliary transport and, upon reaching the microdialysis probe, diffuses across the dialysis membrane and is collected in the dialysate leaving the probe. The rate of MCT is calculated from the length of time from dye deposition to recovery. The rate of tracheal MCT in normal mice was 2.2 +/- 0.45 (SE) mm/min (n = 6), a value similar to that in reports using other techniques. MCT in CF mice was not different (2.3 +/- 0.29, n = 6), consistent with previous observations suggesting that tracheal ion transport properties are not different between CF and normal mice. The rate of MCT in the nasal cavity of normal mice was slower than in the trachea (1.3 +/- 0.26, n = 4). MCT in the CF mouse nasal cavity (1.4 +/- 0.31, n = 8), a region in which the CF mouse exhibits bioelectric properties similar to the human CF patient, was, again, not different from the normal mouse, perhaps reflecting copious gland secretion offsetting Na(+) and liquid hyperabsorption. In conclusion, we have developed a versatile, simple in vivo method to measure MCT in both upper and lower airways of mice and larger animals.

Isolation and culture of airway epithelial cells from chronically infected human lungs.

We describe procedures for isolating and culturing airway epithelial cells from chronically infected human lungs. Experience in our laboratory demonstrated the need to balance pathogen eradication against antibiotic toxicity to epithelial cells. To provide a logical basis for antibiotic selection and dose, we systematically analyzed the cytotoxicity of antibiotics useful against typical pathogens. Alone, colistin, ciprofloxacin, doxycycline, and tobramycin were moderately toxic at concentrations close to those used in cell culture, whereas amphotericin, ceftazidime, chloramphenicol, imipenem, meropenem, piperacillin, sulfamethoxazole/trimethoprim, and vancomycin were nontoxic even at concentrations many times the antimicrobial level. Epithelial cytotoxicity of combined antibiotics was additive, with no evidence of competition or synergism. Antibiotics had little effect on initial cell attachment and did not acutely lyse cells, but inhibited subsequent growth. Interestingly, cytotoxicity decreased markedly with increasing epithelial cell density. Cystic fibrosis (CF) and non-CF epithelial cells showed no differences in sensitivity to the antibiotics tested and initial exposure to antibiotics did not affect the electrophysiologic properties of resistance or short circuit current in well-differentiated cells. Tailored combinations of antibiotics at appropriate doses killed even multidrug-resistant bacteria. Thus, epithelial cells can usually be cultured from chronically infected CF airways.

The CF salt controversy: in vivo observations and therapeutic approaches.

There is controversy over whether abnormalities in the salt concentration or volume of airway surface liquid (ASL) initiate cystic fibrosis (CF) airway disease. In vivo studies of CF mouse nasal epithelia revealed an increase in goblet cell number that was associated with decreased ASL volume rather than abnormal [Cl(-)]. Aerosolization of osmolytes in vivo failed to raise ASL volume. In vitro studies revealed that osmolytes and pharmacological agents were effective in producing isotonic volume responses in human airway epithelia but were typically short acting and less effective in CF cultures with prolonged volume hyperabsorption and mucus accumulation. These data show that (1) therapies can be designed to normalize ASL volume, without producing deleterious compositional changes in ASL, and (2) therapeutic efficacy will likely depend on development of long-acting pharmacologic agents and/or an increased efficiency of osmolyte delivery.

The relative roles of passive surface forces and active ion transport in the modulation of airway surface liquid volume and composition.

Two hypotheses have been proposed recently that offer different views on the role of airway surface liquid (ASL) in lung defense. The "compositional" hypothesis predicts that ASL [NaCl] is kept low (<50 mM) by passive forces to permit antimicrobial factors to act as a chemical defense. The "volume" hypothesis predicts that ASL volume (height) is regulated isotonically by active ion transport to maintain efficient mechanical mucus clearance as the primary form of lung defense. To compare these hypotheses, we searched for roles for: (1) passive forces (surface tension, ciliary tip capillarity, Donnan, and nonionic osmolytes) in the regulation of ASL composition; and (2) active ion transport in ASL volume regulation. In primary human tracheobronchial cultures, we found no evidence that a low [NaCl] ASL could be produced by passive forces, or that nonionic osmolytes contributed substantially to ASL osmolality. Instead, we found that active ion transport regulated ASL volume (height), and that feedback existed between the ASL and airway epithelia to govern the rate of ion transport and volume absorption. The mucus layer acted as a "reservoir" to buffer periciliary liquid layer height (7 microm) at a level optimal for mucus transport by donating or accepting liquid to or from the periciliary liquid layer, respectively. These data favor the active ion transport/volume model hypothesis to describe ASL physiology.

Alterations in airway ion transport in NKCC1-deficient mice.

Airways of Na(+)-K(+)-2Cl(-) (NKCC1)-deficient mice (-/-) were studied in Ussing chambers to determine the role of the basolateral NKCC1 in transepithelial anion secretion. The basal short-circuit current (I(sc)) of tracheae and bronchi from adult mice did not differ between NKCC1-/- and normal mice, whereas NKCC1-/- tracheae from neonatal mice exhibited a significantly reduced basal I(sc). In normal mouse tracheae, sensitivity to the NKCC1 inhibitor bumetanide correlated inversely with the age of the mouse. In contrast, tracheae from NKCC1-/- mice at all ages were insensitive to bumetanide. The anion secretory response to forskolin did not differ between normal and NKCC1-/- tissues. However, when larger anion secretory responses were induced with UTP, airways from the NKCC1-/- mice exhibited an attenuated response. Ion substitution and drug treatment protocols suggested that HCO secretion compensated for reduced Cl(-) secretion in NKCC1-/- airway epithelia. The absence of spontaneous airway disease or pathology in airways from the NKCC1-/- mice suggests that the NKCC1 mutant mice are able to compensate adequately for absence of the NKCC1 protein.

Cloning and functional characterization of two murine uridine nucleotide receptors reveal a potential target for correcting ion transport deficiency in cystic fibrosis gallbladder.

Extracellular nucleotides regulate transepithelial ion secretion via multiple receptors. The P2Y(2) receptor is the predominant transducer of chloride transport responses to nucleotides in the airways, but the P2 receptors that control ion transport in gastrointestinal epithelia have not been identified. UTP and UDP promote chloride secretion in mouse jejuna and gallbladder epithelia, respectively, and these responses were unaffected by P2Y(2) receptor gene disruption. Pharmacological data suggested the involvement of P2Y(4) and P2Y(6) receptors in gastrointestinal responses. To identify the P2Y receptors responsible for the gastrointestinal actions of UTP and UDP, we have cloned the murine P2Y(4) and P2Y(6) receptors and have stably expressed each in a null cell line to examine the nucleotide-promoted inositol phosphate formation and intracellular Ca(2+) mobilization. The (m)P2Y(4) receptor was potently, but not selectively, activated by UTP (UTP > or = ATP >ITP > GTP > CTP), and it was not activated by UDP or ADP. The (m)P2Y(6) receptor was highly selective for UDP (UDP >> ADP = GDP). The nucleotide selectivities observed with the recombinant (m)P2Y(4) and (m)P2Y(6) receptors resemble those for nucleotide-promoted chloride transport in murine P2Y(2)(-/-) jejuna and gallbladder epithelial cells, respectively. Ion transport responses to nucleotide additions were examined in freshly excised tissues from cystic fibrosis transmembrane regulator-deficient mice. Although the effect of UTP on jejunal short-circuit current (I(sc)) was impaired in the CF mouse, UDP-promoted I(sc) changes were not affected in CF gallbladder epithelium, suggesting that the P2Y(6) receptor is a target for treatment of CF gallbladder disease.

Intestinal ion transport in NKCC1-deficient mice.

The Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) located on the basolateral membrane of intestinal epithelia has been postulated to be the major basolateral Cl(-) entry pathway. With targeted mutagenesis, mice deficient in the NKCC1 protein were generated. The basal short-circuit current did not differ between normal and NKCC1 -/- jejuna. In the -/- jejuna, the forskolin response (22 microA/cm(2); bumetanide insensitive) was significantly attenuated compared with the bumetanide-sensitive response (52 microA/cm(2)) in normal tissue. Ion-replacement studies demonstrated that the forskolin response in the NKCC1 -/- jejuna was HCO(3)(-) dependent, whereas in the normal jejuna it was independent of the HCO(3)(-) concentration in the buffer. NKCC1 -/- ceca exhibited a forskolin response that did not differ significantly from that of normal ceca, but unlike that of normal ceca, was bumetanide insensitive. Ion-substitution studies suggested that basolateral HCO(3)(-) as well as Cl(-) entry (via non-NKCC1) paths played a role in the NKCC1 -/- secretory response. In contrast to cystic fibrosis mice, which lack both basal and stimulated Cl(-) secretion and exhibit severe intestinal pathology, the absence of intestinal pathology in NKCC1 -/- mice likely reflects the ability of the intestine to secrete HCO(3)(-) and Cl(-) by basolateral entry mechanisms independent of NKCC1.

Effect of loss of P2Y(2) receptor gene expression on nucleotide regulation of murine epithelial Cl(-) transport.

Extracellular nucleotides are believed to be important regulators of ion transport in epithelial tissues as a result of their ability to activate cell surface receptors. Although numerous receptors that bind nucleotides have been identified, the complexity of this receptor family, combined with the lack of pharmacological agents specific for these receptors, has made the assignment of particular receptors and ligands to physiological responses difficult. Because ATP and UTP appear equipotent and equieffective in regulating ion transport in many epithelia, we tested the hypothesis that the P2Y(2) receptor (P2Y(2)-R) subtype mediates these responses in mouse epithelia, with gene targeting techniques. Mice with the P2Y(2)-R locus targeted and inactivated (P2Y(2)-R(-/-)) were generated, airways (trachea), gallbladder, and intestines (jejunum) excised, and Cl(-) secretory responses to luminal nucleotide additions measured in Ussing chambers. Comparison of P2Y(2)-R(+/+) with P2Y(2)-R(-/-) mice revealed that P2Y(2)-R mediated most (>85-95%) nucleotide-stimulated Cl(-) secretion in trachea, about 50% of nucleotide responses in the gallbladder, and none of the responses in the jejunum. Dose-effect relationships for nucleotides in tissues from P2Y(2)-R(-/-) mice suggest that the P2Y(6)-R regulates ion transport in gallbladder and to a lesser extent trachea, whereas P2Y(4) and/or unidentified receptor(s) regulate ion transport in jejunum. We conclude that the P2Y(2) receptor is the dominant P2Y purinoceptor that regulates airway epithelial ion transport, whereas other P2Y receptor subtypes are relatively more important in other nonrespiratory epithelia.

Ion transport across the normal and CF neonatal murine intestine.

Neonatal mice with cystic fibrosis (CF) exhibit a very high mortality due to intestinal obstruction localized primarily to the ileum and colon. It has been hypothesized that lack of Cl(-) secretion and possibly elevated Na(+) absorption contribute to the gut problems in CF neonates. Therefore, intestines (ileum, proximal colon, and distal colon) from normal and CF day-old mouse pups were studied on ultra-small-aperture (0.0135 cm(2)) Ussing chambers. All three regions of the normal neonatal intestine responded to forskolin with an increase in short-circuit current, which was completely absent in the CF intestine. The neonatal distal colon exhibited a high rate of amiloride-sensitive electrogenic Na(+) absorption, which did not differ between the normal and CF preparations. The ileum and proximal colon of both genotypes exhibited a small but significant electrogenic Na(+) absorption. The neonatal proximal colon and ileum also exhibited electrogenic Na(+)-glucose cotransport, which was significantly greater in the normal compared with the CF ileum. In addition, all three intestinal regions exhibited electrogenic Na(+)-alanine cotransport, which was significantly reduced in two of the regions of the CF neonatal intestine. It is speculated that: 1) the reduced rate of Na(+)-nutrient cotransport in the CF intestine contributes to the lower rate of growth in CF pups, whereas 2) the elevated electrogenic Na(+) absorption in the neonatal intestine, coupled with an inability to secrete Cl(-), contributes to the intestinal obstruction in the CF pups.

Salt restriction induces pseudohypoaldosteronism type 1 in mice expressing low levels of the beta-subunit of the amiloride-sensitive epithelial sodium channel.

The amiloride-sensitive epithelial sodium channel (ENaC) is a heteromultimer of three homologous subunits (alpha-, beta-, and gamma-subunits). To study the role of the beta-subunit in vivo, we analyzed mice in which the betaENaC gene locus was disrupted. These mice showed low levels of betaENaC mRNA expression in kidney (approximately 1%), lung (approximately 1%), and colon (approximately 4%). In homozygous mutant betaENaC mice, no betaENaC protein could be detected with immunofluorescent staining. At birth, there was a small delay in lung-liquid clearance that paralleled diminished amiloride-sensitive Na+ absorption in tracheal explants. With normal salt intake, these mice showed a normal growth rate. However, in vivo, adult betaENaC m/m mice exhibited a significantly reduced ENaC activity in colon and elevated plasma aldosterone levels, suggesting hypovolemia and pseudohypoaldosteronism type 1. This phenotype was clinically silent, as betaENaC m/m mice showed no weight loss, normal plasma Na+ and K+ concentrations, normal blood pressure, and a compensated metabolic acidosis. On low-salt diets, betaENaC-mutant mice developed clinical symptoms of an acute pseudohypoaldosteronism type 1 (weight loss, hyperkalemia, and decreased blood pressure), indicating that betaENaC is required for Na+ conservation during salt deprivation.

Pathophysiology of gene-targeted mouse models for cystic fibrosis.

Pathophysiology of Gene-Targeted Mouse Models for Cystic Fibrosis. Physiol. Rev. 79, Suppl.: S193-S214, 1999. - Mutations in the gene causing the fatal disease cystic fibrosis (CF) result in abnormal transport of several ions across a number of epithelial tissues. In just 3 years after this gene was cloned, the first CF mouse models were generated. The CF mouse models generated to date have provided a wealth of information on the pathophysiology of the disease in a variety of organs. Heterogeneity of disease in the mouse models is due to the variety of gene-targeting strategies used in the generation of the CF mouse models as well as the diversity of the murine genetic background. This paper reviews the pathophysiology in the tissues and organs (gastrointestinal, airway, hepatobiliary, pancreas, reproductive, and salivary tissue) involved in the disease in the various CF mouse models. Marked similarities to and differences from the human disease have been observed in the various murine models. Some of the CF mouse models accurately reflect the ion-transport abnormalities and disease phenotype seen in human CF patients, especially in gastrointestinal tissue. However, alterations in airway ion transport, which lead to the devastating lung disease in CF patients, appear to be largely absent in the CF mouse models. Reasons for these unexpected findings are discussed. This paper also reviews pharmacotherapeutic and gene therapeutic studies in the various mouse models.

Effect of in vivo corticosteroids on Na+ transport across airway epithelia.

We have investigated the role in vivo of mineralocorticoid and glucocorticoid hormones in regulating the rate of electrogenic amiloride-sensitive Na+ absorption across murine airway tissue studied in vivo (nasal potential difference) and in vitro (Ussing chambers). We found that elevating the plasma aldosterone concentration 10-fold (low-Na+ diet) had no significant effect on amiloride-sensitive Na+ absorption across tracheal or nasal epithelia. High doses of dexamethasone for 1 wk likewise did not change the rate of amiloride-sensitive Na+ absorption across airway epithelia. In contrast, both hormonal manipulations elevated the rate of colonic Na+ absorption. Furthermore, adrenalectomy (both normal and cystic fibrosis mice) also failed to alter Na+ absorption across airway epithelia. We conclude that, in vivo, neither the mineralocorticoid nor the glucocorticoid hormones significantly regulate the rates of amiloride-sensitive electrogenic Na+ absorption across airway epithelia in the adult mouse.

Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease.

The pathogenesis of cystic fibrosis (CF) airways infection is unknown. Two hypotheses, "hypotonic [low salt]/defensin" and "isotonic volume transport/mucus clearance," attempt to link defects in cystic fibrosis transmembrane conductance regulator-mediated ion transport to CF airways disease. We tested these hypotheses with planar and cylindrical culture models and found no evidence that the liquids lining airway surfaces were hypotonic or that salt concentrations differed between CF and normal cultures. In contrast, CF airway epithelia exhibited abnormally high rates of airway surface liquid absorption, which depleted the periciliary liquid layer and abolished mucus transport. The failure to clear thickened mucus from airway surfaces likely initiates CF airways infection. These data indicate that therapy for CF lung disease should not be directed at modulation of ionic composition, but rather at restoring volume (salt and water) on airway surfaces.

Enhanced in vivo airway gene transfer via transient modification of host barrier properties with a surface-active agent.

Effective adenoviral gene therapy requires efficient viral vector entry into epithelial cells. Injured airway epithelia display enhanced gene transfer, reflecting in part increased vector access to protected cell populations and/or protected basolateral membranes. We tested whether adenoviral gene transfer is enhanced by modification of the epithelial barrier in mouse nasal airways with a nonionic detergent (polidocanol, PDOC). In C57BL/6 mice, 1.6 x 10(9) PFU of Ad5CMV LacZ (AdLacZ) instilled into the right nostril produced negligible gene transfer to the nasal epithelium 2 days after dosing, but significant, dose-dependent increases in gene transfer were achieved by pretreatment with PDOC. Permeation of the electron-dense tracer lanthanum into the intercellular junctions of PDOC (0.1%)-treated murine nasal epithelium, but not into intercellular junctions of vehicle controls, is consistent with PDOC-mediated increases in tight junctional permeability. In CF(-/-) mice, significant gene expression was not detectable after exposure to Ad5CBCFTR alone (1.4 x 10(9) PFU in 20 microl; AdCFTR), but PDOC pretreatment prior to AdCFTR instillation produced functional expression of CFTR (measured as deltaPD) 5 days after instillation. Because the development and testing of lung gene therapy will principally occur in children and adults with airway disease, AdLacZ gene transfer with and without PDOC pretreatment was examined in infected nasal airways. Gene expression was significantly reduced in infected as compared with uninfected airways. We conclude that the use of adjuvant surface-active and/or membrane-perturbing agents, synthetic or naturally derived, may provide a novel approach to enhancing the efficiency of adenoviral gene transfer.