Larry G. Johnson

A Controlled Study of Adenoviral-Vector–Mediated Gene Transfer in the Nasal Epithelium of Patients with Cystic Fibrosis

BACKGROUND Cystic fibrosis is a monogenic disease that deranges multiple systems of ion transport in the airways, culminating in chronic infection and destruction of the lung. The introduction of a normal copy of the cystic fibrosis transmembrane conductance regulator (CFTR) gene into the airway epithelium through gene transfer is an attractive approach to correcting the underlying defects in patients with cystic fibrosis. We tested the feasibility of gene therapy using adenoviral vectors in the nasal epithelium of such patients. METHODS An adenoviral vector containing the normal CFTR complementary DNA in four logarithmically increasing doses (estimated multiplicity of infection, 1, 10, 100, and 1000), or vehicle alone, was administered in a randomized, blinded fashion to the nasal epithelium of 12 patients with cystic fibrosis. Gene transfer was quantitated by molecular techniques that detected the expression of CFTR messenger RNA and by functional measurements of transepithelial potential differences (PDs) to assess abnormalities of ion transport specific to cystic fibrosis. The safety of this treatment was monitored by nasal lavage and biopsy to assess inflammation and vector replication. RESULTS The adenoviral vector was detected in nasal-lavage fluid by culture, the polymerase chain reaction (PCR), or both in a dose-dependent fashion for up to eight days after vector administration. There was molecular evidence of gene transfer by reverse-transcriptase PCR assays or in situ hybridization in five of six patients treated at the two highest doses. However, the percentage of epithelial cells transfected by the vector was very low (< 1 percent), and measurement of PD across the epithelium revealed no significant restoration of chloride transport or normalization of sodium transport. At the lower doses of vector, there were no toxic effects. However, at the highest dose there was mucosal inflammation in two of three patients. CONCLUSIONS In patients with cystic fibrosis, adenoviral-vector-mediated transfer of the CFTR gene did not correct functional defects in nasal epithelium, and local inflammatory responses limited the dose of adenovirus that could be administered to overcome the inefficiency of gene transfer.

Role of Mutant CFTR in Hypersusceptibility of Cystic Fibrosis Patients to Lung Infections

Cystic fibrosis (CF) patients are hypersusceptible to chronic Pseudomonas aeruginosa lung infections. Cultured human airway epithelial cells expressing the ΔF508 allele of the cystic fibrosis transmembrane conductance regulator (CFTR) were defective in uptake of P. aeruginosa compared with cells expressing the wild-type allele. Pseudomonas aeruginosa lipopolysaccharide (LPS)-core oligosaccharide was identified as the bacterial ligand for epithelial cell ingestion; exogenous oligosaccharide inhibited bacterial ingestion in a neonatal mouse model, resulting in increased amounts of bacteria in the lungs. CFTR may contribute to a host-defense mechanism that is important for clearance of P. aeruginosa from the respiratory tract.

Abnormal surface liquid pH regulation by cultured cystic fibrosis bronchial epithelium.

Cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-dependent airway epithelial bicarbonate transport is hypothesized to participate in airway surface liquid pH regulation and contribute to lung defense. We measured pH and ionic composition in apical surface liquid (ASL) on polarized normal (NL) and CF primary bronchial epithelial cell cultures under basal conditions, after cAMP stimulation, and after challenge with luminal acid loads. Under basal conditions, CF epithelia acidified ASL more rapidly than NL epithelia. Two ASL pH regulatory paths that contributed to basal pH were identified in the apical membrane of airway epithelia, and their activities were measured. We detected a ouabain-sensitive (nongastric) H+,K+-ATPase that acidified ASL, but its activity was not different in NL and CF cultures. We also detected the following evidence for a CFTR-dependent \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document} secretory pathway that was defective in CF: (i) ASL [\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document}] was higher in NL than CF ASL; (ii) activating CFTR with forskolin/3-isobutyl-1-methylxanthine alkalinized NL ASL but acidified CF ASL; and (iii) NL airway epithelia more rapidly and effectively alkalinized ASL in response to a luminal acid challenge than CF epithelia. We conclude that cultured human CF bronchial epithelial pHASL is abnormally regulated under basal conditions because of absent CFTR-dependent \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document} secretion and that this defect can lead to an impaired capacity to respond to airway conditions associated with acidification of ASL.

Loss of binding and entry of liposome-DNA complexes decreases transfection efficiency in differentiated airway epithelial cells.

The target cells for gene therapy of cystic fibrosis lung disease are the well differentiated cells that line airway lumens. Employing cultures of airway epithelial cells that grow like “islands” and exhibit a continuum of cellular differentiation, we studied the mechanisms that render well differentiated cells more difficult to transfect with cationic liposomes than poorly differentiated cells. The poorly differentiated cells at the edge of the islands were transfectable with liposome-DNA complexes (pCMVβ:LipofectACE = 1:5 (w/w)), whereas the more differentiated cells in the center of the islands were not. Evaluation of the steps leading to lipid-mediated transfection revealed that edge cells bound more liposome-DNA complexes, in part due to a more negative surface charge (as measured by cationized ferritin binding), and that edge cells internalized more liposome-DNA complexes than central cells. Edge cells exhibited receptor-mediated endocytosis of LDL, pinocytosis of 10-nm microspheres, and phagocytosis of 2-μm microspheres, whereas central cells were only capable of receptor-mediated endocytosis. Cytochalasin B, which inhibited pinocytosis by 65% and phagocytosis by 93%, decreased edge cell liposome-DNA complex entry by 50%. Potassium depletion, which decreased phagocytosis by >90% but had no effect on pinocytosis, inhibited edge cell liposome-DNA complex entry by 71%. These results indicate that liposome-DNA complexes enter edge cells via phagocytosis and that this pathway is not detectable in central cells. In conclusion, both reduced negative surface charge and absence of phagocytosis internalization pathways in relatively differentiated cells may explain differentiation-dependent decrements in cationic liposome-mediated gene transfer in airway epithelia.

Normalization of raised sodium absorption and raised calcium-mediated chloride secretion by adenovirus-mediated expression of cystic fibrosis transmembrane conductance regulator in primary human cystic fibrosis airway epithelial cells.

Cystic fibrosis airway epithelia exhibit a spectrum of ion transport properties that differ from normal, including not only defective cAMP-mediated Cl- secretion, but also increased Na+ absorption and increased Ca(2+)-mediated Cl- secretion. In the present study, we examined whether adenovirus-mediated (Ad5) transduction of CFTR can correct all of these CF ion transport abnormalities. Polarized primary cultures of human CF and normal nasal epithelial cells were infected with Ad5-CBCFTR at an moi (10(4)) which transduced virtually all cells or Ad5-CMV lacZ as a control. Consistent with previous reports, Ad5-CBCFTR, but not Ad5-CMV lacZ, corrected defective CF cAMP-mediated Cl- secretion. Basal Na+ transport rates (basal Ieq) in CF airway epithelial sheets (-78.5 +/- 9.8 microA/cm2) were reduced to levels measured in normal epithelial sheets (-30.0 +/- 2.0 microA/cm2) by Ad5-CBCFTR (-36.9 +/- 4.8 microA/cm2), but not Ad5-CMV lacZ (-65.8 +/- 6.1 microA/cm2). Surprisingly, a significant reduction in delta Ieq in response to ionomycin, a measure of Ca(2+)-mediated Cl- secretion, was observed in CFTR-expressing (corrected) CF epithelial sheets (-6.9 +/- 11.8 microA/cm2) when compared to uninfected CF epithelial sheets (-76.2 +/- 15.1 microA/cm2). Dose response effects of Ad5-CBCFTR on basal Na+ transport rates and Ca(2+)-mediated Cl- secretion suggest that the mechanism of regulation of these two ion transport functions by CFTR may be different. In conclusion, efficient transduction of CFTR corrects hyperabsorption of Na+ in primary CF airway epithelial cells and restores Ca(2+)-mediated Cl- secretion to levels observed in normal airway epithelial cells. Moreover, assessment of these ion transport abnormalities may represent important endpoints for testing the efficacy of gene therapy for cystic fibrosis.

Pseudotyped human lentiviral vector-mediated gene transfer to airway epithelia in vivo

We used a replication defective human lentiviral (HIV) vector encoding the lacZ cDNA and pseudotyped with the vesicular stomatitis virus (VSV) glycoprotein (G) to evaluate the utility of this vector system in airway epithelia. In initial studies, apical application of vector to polarized well differentiated human airway epithelial cell cultures produced minimal levels of transgene expression whereas basolateral application of vector enhanced levels of transduction approximately 30-fold. Direct in vivo delivery of HIV vectors to the nasal epithelium and tracheas of mice failed to mediate gene transfer, but injury with sulfur dioxide (SO2) before vector delivery enhanced gene transfer efficiency to the nasal epithelium of both mice and rats. SO2 injury also enhanced HIV vector-mediated gene transfer to the tracheas of rodents. These data suggest that SO2 injury increases access of vector to basal cells and/or the basolateral membrane of airway surface epithelial cells. Quantification of gene transfer efficiency in murine tracheas demonstrated that transduction was more efficient when vector was delivered on the day of exposure (7.0%, n = 4) than when vector was delivered on the day after SO2 exposure (1.7%, n = 4).

Influenza M2 envelope protein augments avian influenza hemagglutinin pseudotyping of lentiviral vectors

Lentivirus-based gene transfer has the potential to efficiently deliver DNA-based therapies into non-dividing epithelial cells of the airway for the treatment of lung diseases such as cystic fibrosis. However, significant barriers both to lung-specific gene transfer and to production of lentivirus vectors must be overcome before these vectors can be routinely used for applications to the lung. In this study, we investigated whether the ability to produce lentiviral vectors pseudotyped with fowl plague virus hemagglutinin (HA) could be improved by co-expression of influenza virus M2 in vector-producing cells. We found that M2 expression led to a 10–30-fold increase in production of HA-pseudotyped lentivirus vectors based upon equine infectious anemia virus (EIAV) or human immunodeficiency virus type 1 (HIV-1). Experiments using the M2 inhibitor amantadine and a drug-resistant mutant of M2 established that the ion channel activity of M2 was important for M2-dependent augmentation of vector production. Furthermore, the neuraminidase activity necessary for particle release from producer cells could also be incorporated into producer cells by co-expression of influenza NA cDNA. Lentiviral vectors pseudotyped with influenza envelope proteins were able to efficiently transduce via the apical membrane of polarized mouse tracheal cultures in vitro as well as mouse tracheal epithelia in vivo.

Retroviral Approaches to Gene Therapy of Cystic Fibrosis

Abstract: Retroviral vectors are attractive as vectors for gene therapy of cystic fibrosis because of their ability to integrate into the host cell genome, which may lead to long‐term expression and, perhaps, a cure. Nevertheless, retroviral applications for gene transfer to airway epithelia have been limited by low titers and a requirement for proliferating cells. Significant advances in pseudotyping of retroviruses and in retroviral production have reduced some of the concerns regarding titer. The development of lentiviral vectors that transduce nondividing cells has also helped to establish that retroviral approaches for gene therapy of cystic fibrosis are feasible. However, the apical membrane of the airway epithelium remains a formidable barrier to gene transfer. In this review, I will discuss limitations of current retroviral gene transfer vectors and strategies to improve retroviral gene transfer efficiency to airway epithelia in vivo.

Attenuation of monocrotaline-induced pulmonary hypertension by luminal adeno-associated virus serotype 9 gene transfer of prostacyclin synthase.

Idiopathic pulmonary arterial hypertension (iPAH) is associated with high morbidity and mortality. We evaluated whether luminal delivery of the human prostacyclin synthase (hPGIS) cDNA with adeno-associated virus (AAV) vectors could attenuate PAH. AAV serotype 5 (AAV5) and AAV9 vectors containing the hPGIS cDNA under the control of a cytomegalovirus-enhanced chicken β-actin (CB) promoter or vehicle (saline) were instilled into lungs of rats. Two days later, rats were injected with monocrotaline (MCT, 60 mg/kg) or saline. Biochemical, hemodynamic, and morphologic assessments were performed when the rats developed symptoms (3-4 weeks) or at 6 weeks. Luminal (airway) administration of AAV5 and AAV9CBhPGIS vectors (MCT-AAV5 and MCT-AAV9 rats) significantly increased plasma levels of 6-keto-PGF1(α) as compared with MCT-controls, and closely resembled levels measured in rats not treated with MCT (saline-saline). Right ventricular (RV)/left ventricular (LV)+septum (S) ratios and RV systolic pressure (RVSP) were greater in MCT-control rats than in saline-saline rats, whereas the ratios and RVSP in MCT-AAV5CBhPGIS and MCT-AAV9CBhPGIS rats were similar to saline-saline rats. Thickening of the muscular media of small pulmonary arteries of MCT-control rats was detected in histological sections, whereas the thickness of the muscular media in MCT-AAV5CBhPGIS and MCT-AAV9CBhPGIS rats was similar to saline-saline controls. In experiments with different promoters, a trend toward increased levels of PGF1(α) expression was detected in lung homogenates, but not plasma, of MCT-treated rats transduced with an AAV9-hPGIS vector containing a CB promoter. This correlated with significant reductions in the RV/LV+S ratio and RVSP in MCT-AAV9CBhPGIS rats that resembled levels in saline-saline rats. No changes in levels of PGF1(α), RV/LV+S, or RVSP were detected in rats transduced with AAV9-hPGIS vectors containing a modified CB promoter (CB7) or a distal epithelial cell-specific promoter (CC10). Thus, AAV9CBhPGIS vectors prevented development of MCT-induced PAH and associated pulmonary vascular remodeling.

Macromolecular Transport across Nasal and Respiratory Epithelia

The airway and alveolar epithelia in conjunction with the mucociliary escalator act as a barrier to prevent the translocation of inhaled macromolecules, e.g., protein and peptide antigens, across pulmonary surfaces. Nevertheless, some exogenous macromolecules appear to cross the epithelia antigenically intact through either the paracellular or the transcellular route. Endogenous macromolecules including albumin, immunoglobulin, lactoferrin, α2-macroglobin, and α1antitrypsin have been measured in either bronchoalveolar lavage fluid or the liquid that lines airway surfaces, airway surface liquid (Mentz et al., 1984; Bignon et al., 1976). These proteins may have specialized methods for translocation into and out of the airway surface liquid and may also play significant roles in fluid homeostasis, particularly in the alveolar region. Yet the knowledge of the mechanisms responsible for the maintenance of protein gradients across respiratory epithelia is primitive in relation to similar knowledge regarding ion gradients.