Recovery of airway cystic fibrosis transmembrane conductance regulator function in mice with cystic fibrosis after single-dose lentivirus-mediated gene transfer

The potential for gene therapy to be an effective treatment for cystic fibrosis (CF) airway disease has been limited by inefficient gene transfer vector particle delivery and lack of persistent gene expression. We have developed an airway conditioning process that, when combined with a human immunodeficiency virus (HIV)-derived lentivirus (LV) vector, resulted in persistent in vivo expression of transgenes in airway epithelium. Pretreatment of mouse nasal epithelium with the detergent lysophosphatidylcholine (LPC) prior to instillation of a single dose of an LV vector carrying the LacZ marker gene produced significant LacZ gene expression in nasal airway epithelium for at least 92 days. Transduction of the cystic fibrosis transmembrane conductance regulator (CFTR) gene using the same LV vector system resulted in partial recovery of electrophysiologic function in the nasal airway epithelium of CF mice (cftr(tm1Unc) knockout) for at least 110 days. This first demonstration of LV-mediated in vivo recovery of CFTR function in CF airway epithelium illustrates the potential of combining a preconditioning of the airway surface with a simple and brief LV vector exposure to produce therapeutic gene expression in airway.     

Genetic modification of adeno-associated viral vector type 2 capsid enhances gene transfer efficiency in polarized human airway epithelial cells

Cystic fibrosis (CF) is a common genetic disease characterized by defects in the expression of the CF transmembrane conductance regulator (CFTR) gene. Gene therapy offers better hope for the treatment of CF. Adeno-associated viral (AAV) vectors are capable of stable expression with low immunogenicity. Despite their potential in CF gene therapy, gene transfer efficiency by AAV is limited because of pathophysiological barriers in these patients. Although a few AAV serotypes have shown better transduction compared with the AAV2-based vectors, gene transfer efficiency in human airway epithelium has still not reached therapeutic levels. To engineer better AAV vectors for enhanced gene delivery in human airway epithelium, we developed and characterized mutant AAV vectors by genetic capsid modification, modeling the well-characterized AAV2 serotype. We genetically incorporated putative high-affinity peptide ligands to human airway epithelium on the GH loop region of AAV2 capsid protein. Six independent mutant AAV were constructed, containing peptide ligands previously reported to bind with high affinity for known and unknown receptors on human airway epithelial cells. The vectors were tested on nonairway cells and nonpolarized and polarized human airway epithelial cells for enhanced infectivity. One of the mutant vectors, with the peptide sequence THALWHT, not only showed the highest transduction in undifferentiated human airway epithelial cells but also indicated significant transduction in polarized cells. Interestingly, this modified vector was also able to infect cells independently of the heparan sulfate proteoglycan receptor. Incorporation of this ligand on other AAV serotypes, which have shown improved gene transfer efficiency in the human airway epithelium, may enhance the application of AAV vectors in CF gene therapy.     

Sendai virus-mediated CFTR gene transfer to the airway epithelium

The potential for gene therapy to be an effective treatment for cystic fibrosis has been hampered by the limited gene transfer efficiency of current vectors. We have shown that recombinant Sendai virus (SeV) is highly efficient in mediating gene transfer to differentiated airway epithelial cells, because of its capacity to overcome the intra- and extracellular barriers known to limit gene delivery. Here, we have identified a novel method to allow the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA sequence to be inserted within SeV (SeV-CFTR). Following in vitro transduction with SeV-CFTR, a chloride-selective current was observed using whole-cell and single-channel patch-clamp techniques. SeV-CFTR administration to the nasal epithelium of cystic fibrosis (CF) mice (Cftr(G551D) and Cftr(tm1Unc)TgN(FABPCFTR)#Jaw mice) led to partial correction of the CF chloride transport defect. In addition, when compared to a SeV control vector, a higher degree of inflammation and epithelial damage was found in the nasal epithelium of mice treated with SeV-CFTR. Second-generation transmission-incompetent F-deleted SeV-CFTR led to similar correction of the CF chloride transport defect in vivo as first-generation transmission-competent vectors. Further modifications to the vector or the host may make it easier to translate these studies into clinical trials of cystic fibrosis.     

Human immunodeficiency viral vector pseudotyped with the spike envelope of severe acute respiratory syndrome coronavirus transduces human airway epithelial cells and dendritic cells

The human severe acute respiratory syndrome coronavirus (SARS-CoV) is a highly infectious virus that causes severe respiratory infections in humans. The spike envelope glycoprotein of SARS-CoV, the main determinant of SARS-CoV tropism, was isolated and used to pseudotype a human immunodeficiency virus (HIV)-based vector. Spike-pseudotyped HIV vector was generated and evaluated in vitro on well-differentiated human airway epithelial cells and bronchial explants and in vivo in murine airways. The spike envelope was less efficient at promoting HIV vector transduction of murine airway epithelium than an optimized deletion mutant of the Zaire ebolavirus envelope glycoprotein (NTD6L), which was used as a benchmark. However, spike-pseudotyped HIV vector was substantially more efficient than NTD6L-pseudotyped vector on human airway epithelium as demonstrated by lacZ gene transfer in primary cultures of epithelial cells and bronchial explants. In addition, this study shows that spike-pseudotyped HIV -based vector can efficiently transduce human dendritic cells and epithelial cells of the esophagus, which may have implications in investigating mechanisms of SARS-CoV pathogenesis. Spike-pseudotyped HIV-based vector is a novel lung-directed gene transfer vehicle that holds promise for the treatment of genetic lung diseases such as cystic fibrosis or alpha(1)-antitrypsin deficiency.     

A phase I study of adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator gene to a lung segment of individuals with cystic fibrosis

A third-generation adenoviral vector containing recombinant human cystic fibrosis transmembrane conductance regulator (CFTR) gene was delivered by bronchoscope in escalating doses to the conducting airway of 11 volunteers with cystic fibrosis. Assessments of dose-limiting toxicity (DLT), efficiency of gene transfer, and cell-mediated and humoral immune responses to vector administration were performed. DLT, manifest by flulike symptoms and transient radiographic infiltrates, was seen at 2.1 x 10(11) total viral particles. A highly specific assay for gene transfer was developed using in situ hybridization with an oligoprobe against unique vector sequence. Detectable gene transfer was observed in harvested bronchial epithelial cells (<1%) 4 days after vector instillation, which diminished to undetectable levels by day 43. Adenovirus-specific cell-mediated T cells were induced in most subjects, although only mild increases in systemic humoral immune response were observed. These results demonstrate that gene transfer to epithelium of the lower respiratory tract can be achieved in humans with adenoviral vectors but that efficiency is low and of short duration in the native CF airway.     

Targeting viral-mediated transduction to the lung airway epithelium with the anti-inflammatory cationic lipid dexamethasone-spermine.

We formulated adenovirus (AdV) vectors with cationic steroid liposomes containing dexamethasone-spermine (DS)/dioleoylphosphatidylethanolamine (DOPE) in an effort to overcome the lack of apically expressed AdV vector receptors on airway epithelial cells and to reduce the inflammation associated with AdV vector exposure. An AdV vector (1 to 2.5 x 10(11) genome copies) expressing human placental alkaline phosphatase or beta-galactosidase (LacZ) was delivered alone or complexed with DS/DOPE, DC-Chol/DOPE, or dexamethasone to C57Bl/6 mice via intranasal instillation. Formulation of the AdV vector with DS/DOPE and DC-Chol/DOPE resulted in transgene expression targeted only to the airway epithelial cells with minimal expression in alveolar cells, while AdV alone caused high alveolar transduction. The DS/DOPE and dexamethasone formulations greatly reduced cellular infiltrates compared to AdV vector alone, while formulation with DC-Chol/DOPE did not. IFN-gamma was significantly elevated at day 7 in mice receiving only the AdV vector compared to the AdV vector formulated with DS/DOPE, DC-Chol/DOPE, or dexamethasone. Lipid formulation of adeno-associated virus vector expressing LacZ also produced airway epithelial targeting, similar to the AdV vector. Viral vectors can be formulated with DS/DOPE to improve targeting to the airway epithelium in vivo and to attenuate vector-induced inflammation through the pharmacological activity of DS.     

Endosomal processing limits gene transfer to polarized airway epithelia by adeno-associated virus

The restriction of viral receptors and coreceptors to the basolateral surface of airway epithelial cells has been blamed for the inefficient transfer of viral vectors to the apical surface of this tissue. We now report, however, that differentiated human airway epithelia internalize rAAV type-2 virus efficiently from their apical surfaces, despite the absence of known adeno-associated virus-2 (AAV-2) receptors or coreceptors at these sites. The dramatically lower transduction efficiency of rAAV infection from the apical surface of airway cells appears to result instead from differences in endosomal processing and nuclear trafficking of apically or basolaterally internalized virions. AAV capsid proteins are ubiquitinated after endocytosis, and gene transfer can be significantly enhanced by proteasome or ubiquitin ligase inhibitors. Tripeptide proteasome inhibitors increased persistent rAAV gene delivery from the apical surface >200-fold, to a level nearly equivalent to that achieved with basolateral infection. In vivo application of proteasome inhibitor in mouse lung augmented rAAV gene transfer from undetectable levels to a mean of 10.4 +/- 1.6% of the epithelial cells in large bronchioles. Proteasome inhibitors also increased rAAV-2-mediated gene transfer to the liver tenfold, but they did not affect transduction of skeletal or cardiac muscle. These findings suggest that tissue-specific ubiquitination of viral capsid proteins interferes with rAAV-2 transduction and provides new approaches to circumvent this barrier for gene therapy of diseases such as cystic fibrosis.     

In vivo gene transfer to the mouse eye using an HIV-based lentiviral vector; efficient long-term transduction of corneal endothelium and retinal pigment epithelium

We have evaluated the transduction profiles of an HIV-based lentiviral vector delivered regionally to ocular tissues in vivo. Following subretinal injection, a green fluorescent protein (GFP) reporter gene was efficiently and stably expressed in retinal pigment epithelial (RPE) cells. Limited transduction of adjacent photoreceptors occurred in newborn mice, but was inefficient in adult animals. Injection of the vector into the anterior chamber resulted in efficient and stable transduction of corneal endothelial cells. Efficient in vivo gene transfer into cells of the corneal endothelium and retinal pigment epithelium by lentiviral vectors may therefore offer a valuable approach to the treatment of disorders of the cornea and outer retina.     

A defective nontransmissible recombinant Sendai virus mediates efficient gene transfer to airway epithelium in vivo

Recombinant Sendai virus (SeV)-mediated gene transfer to differentiated airway epithelial cells has shown to be very efficient, because of its ability to overcome the intra- and extracellular barriers known to limit gene delivery. However, this virus is transmission competent and therefore unlikely to be suitable for use in clinical trials. A nontransmissible, replication-competent recombinant SeV has recently been developed by deleting the envelope Fusion (F) protein gene (SeV/DeltaF). Here we show that SeV/DeltaF is able to mediate beta-galactosidase reporter gene transfer to the respiratory tract of mice in vivo, as well as to human nasal epithelial cells in vitro. Further, in an ex vivo model of differentiated airway epithelium, SeV/DeltaF gene transfer was not importantly inhibited by native mucus. When compared to the transmission-competent SeV in vivo, no difference in gene expression was observed at the time of peak expression. The development of an F-defective nontransmissible SeV, which can still efficiently mediate gene transfer to the airway epithelium, represents the first important step towards the use of a cytoplasmic RNA viral vector in clinical trials of gene therapy.     

Activation of Transgene-specific T Cells Following Lentivirus-mediated Gene Delivery to Mouse Lung

Integrating lentiviral vectors based on the human immunodeficiency virus type-1 (HIV-1) can transduce quiescent cells, which in lung account for almost 95% of the epithelial cell population. Pseudotyping lentiviral vectors with the envelope glycoprotein from the Ebola Zaire virus, the lymphocytic choriomeningitis virus (LCMV), the Mokola virus, and the vesicular stomatitis virus (VSV-G) resulted in transduction of mouse alveolar epithelium, but gene expression in the lung of C57BL/6 and BALB/c mice waned within 90 days of vector injection. Intratracheal delivery of the four pseudotyped lentiviral vectors resulted in transgene-specific T-cell activation in both mouse strains, albeit lower than that achieved by intramuscular injection of the vectors. We performed an adoptive transfer of luciferase-specific T cells, isolated from spleen or lung of donor mice injected with VSV-G-pseudotyped lentivirus vector expressing luciferase into the muscle or lung, respectively, into recipient recombination-activating gene (RAG)–deficient mice transduced in lung with adenovirus expressing firefly luciferase (ffluc2). Gene expression declined within 7 days of adoptive transfer approaching background levels by day 36. Taken together, our results suggest that the loss of transduced cells in lung is due to VSV-G.HIV vector–mediated activation of transgene-specific T cells rather than as result of normal turnover of airway cells.     

Lentiviral transduction of the murine lung provides efficient pseudotype and developmental stage-dependent cell-specific transgene expression

Gene transfer for cystic fibrosis (CF) airway disease has been hampered by the lung's innate refractivity to pathogen infection. We hypothesized that early intervention with an integrating gene transfer vector capable of transducing the lung via the lumen may be a successful therapeutic approach. An HIV-based lentiviral vector pseudotyped with the baculovirus gp64 envelope was applied to the fetal, neonatal or adult airways. Fetal intra-amniotic administration resulted in transduction of approximately 14% of airway epithelial cells, including both ciliated and non-ciliated epithelia of the upper, mid and lower airways; there was negligible alveolar or nasal transduction. Following neonatal intra-nasal administration we observed significant transduction of the airway epithelium (approximately 11%), although mainly in the distal lung, and substantial alveolar transduction. This expression was still detectable at 1 year after application. In the adult, the majority of transduction was restricted to the alveoli. In contrast, vesicular stomatitis virus glycoprotein pseudotyped virus transduced only alveoli after adult and neonatal application and no transduction was observed after fetal administration. Repeat administration did not increase transduction levels of the conducting airway epithelia. These data demonstrate that application at early developmental stages in conjunction with an appropriately pseudotyped virus provides efficient, high-level transgene expression in the murine lung. This may provide a modality for treatment for lung disease in CF.     

Repeat administration of an adenovirus vector encoding cystic fibrosis transmembrane conductance regulator to the nasal epithelium of patients with cystic fibrosis.

Cystic fibrosis (CF) is a common autosomal recessive disease caused by mutations in the CF transmembrane conductance regulator gene. Recombinant adenoviruses have shown promise as vectors for transfer of CF transmembrane conductance regulator cDNA to airway epithelia and correction of the Cl- transport defect. However, because adenovirus-mediated gene transfer is transient, use of adenovirus as a vector for treatment of CF would require repeated administration. Therefore, we evaluated repeat administration of an adenovirus vector to the nasal epithelium of patients with CF with five escalating doses of up to 10(10) infectious units. There were no detectable adverse affects. All subjects were initially seropositive but developed additional humoral immune responses. The vector partially corrected the defect in airway epithelial Cl- transport in some subjects, although there was variability between subjects and there was less correction with subsequent administration, perhaps because the immune response limited gene transfer. Future work must focus on vectors with increased efficiency and with the ability to evade host defenses.     

Keratinocyte growth factor gene transduction ameliorates acute lung injury and mortality in mice

At present there is no known effective pharmacological therapy for acute lung injury (ALI). Because keratinocyte growth factor (KGF) promotes epithelial cell growth, intratracheal administration of KGF has the possibility of restoring lung tissue integrity in injured lungs and improving patient outcomes. However, treatment using recombinant KGF protein is limited by its short effective duration. Thus, we investigated the effectiveness of intratracheal KGF gene transduction using adenoviral vector in ALI. We constructed an adenoviral vector expressing mouse KGF (mKGF), and 1.0 x 10(9 ) plaque-forming units of mKGF cDNA-expressing (Ad-KGF) and control (Ad-1w1) adenoviral vector was intratracheally instilled, using a MicroSprayer, into anesthetized BALB/c mice. Three days later, the mice were exposed to >90% oxygen for 72 hr, and the effect of KGF on hyperoxia-induced lung injury was examined. In the Ad-KGF group, KGF was strongly expressed in the airway epithelial cells, while peribronchiolar and alveolar inflammation caused by adenoviral vector instillation was minimal. The KGF overexpression not only induced proliferation of surfactant protein C-positive cuboidal cells, especially in the terminal bronchiolar and alveolar walls, but also prevented lung injury including intraalveolar exudation/hemorrhage, albumin permeability increase, and pulmonary edema. The arterial oxygen tension and the survival rate were significantly higher in the KGF-transfected group. These findings suggest that KGF gene transduction into the airway epithelium is a promising potential treatment for ALI.     

Toward Gene Therapy for Cystic Fibrosis Using a Lentivirus Pseudotyped With Sendai Virus Envelopes

Gene therapy for cystic fibrosis (CF) is making encouraging progress into clinical trials. However, further improvements in transduction efficiency are desired. To develop a novel gene transfer vector that is improved and truly effective for CF gene therapy, a simian immunodeficiency virus (SIV) was pseudotyped with envelope proteins from Sendai virus (SeV), which is known to efficiently transduce unconditioned airway epithelial cells from the apical side. This novel vector was evaluated in mice in vivo and in vitro directed toward CF gene therapy. Here, we show that (i) we can produce relevant titers of an SIV vector pseudotyped with SeV envelope proteins for in vivo use, (ii) this vector can transduce the respiratory epithelium of the murine nose in vivo at levels that may be relevant for clinical benefit in CF, (iii) this can be achieved in a single formulation, and without the need for preconditioning, (iv) expression can last for 15 months, (v) readministration is feasible, (vi) the vector can transduce human air–liquid interface (ALI) cultures, and (vii) functional CF transmembrane conductance regulator (CFTR) chloride channels can be generated in vitro. Our data suggest that this lentiviral vector may provide a step change in airway transduction efficiency relevant to a clinical programme of gene therapy for CF.     

Generation of Novel AAV Variants by Directed Evolution for Improved CFTR Delivery to Human Ciliated Airway Epithelium

Recombinant adeno-associated virus (AAV) vectors expressing the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been used to deliver CFTR to the airway epithelium of cystic fibrosis (CF) patients. However, no significant CFTR function has been demonstrated likely due to low transduction efficiencies of the AAV vectors. To improve AAV transduction efficiency for human airway epithelium (HAE), we generated a chimeric AAV library and performed directed evolution of AAV on an in vitro model of human ciliated airway epithelium. Two independent and novel AAV variants were identified that contained capsid components from AAV-1, AAV-6, and/or AAV-9. The transduction efficiencies of the two novel AAV variants for human ciliated airway epithelium were three times higher than that for AAV-6. The novel variants were then used to deliver CFTR to ciliated airway epithelium from CF patients. Here we show that our novel AAV variants, but not the parental, AAV provide sufficient CFTR delivery to correct the chloride ion transport defect to ~25% levels measured in non-CF cells. These results suggest that directed evolution of AAV on relevant in vitro models will enable further improvements in CFTR gene transfer efficiency and the development of an efficacious and safe gene transfer vector for CF lung disease.     

Cationic Lipid Formulations Alter the In Vivo Tropism of AAV2/9 Vector in Lung

Physicochemical properties of gene transfer vectors play an important role in both transduction efficiency and biodistribution following airway delivery. Adeno-associated virus (AAV) vectors are currently used in many gene transfer applications; however, the respiratory epithelium remains a challenging target. We synthesized two cationic sterol-based lipids, dexamethasone-spermine (DS) and disubstituted spermine (D₂S) for pulmonary gene targeting. Scanning and transmission electron micrographs (TEM) confirmed that AAV/lipid formulations produced submicron-sized clusters. When AAV2/9 or AAV2/6.2 were formulated with these cationic lipids, the complexes had positive zeta potential (ζ) and the transduction efficiency in cultured A549 cells increased by sevenfold and sixfold, respectively. Transduction of cultured human airway epithelium with AAV2/6.2-lipid formulations also showed approximately twofold increase in green fluorescence protein (GFP) positive cells as quantified by flow cytometry. Intranasal administration of 10¹¹ genome copies (GC) of AAV2/9 and AAV2/6.2 coformulated with lipid formulations resulted in an average fourfold increase in transgene expression for both vectors. Formulation of AAV2/9 with DS changed the tropism of this vector for the alveolar epithelium, resulting in successful transduction of conducting airway epithelium. Our results suggest that formulating AAV2/9 and AAV2/6.2 with DS and D₂S can lead to improved physicochemical characteristics for in vivo gene delivery to lung.     

Aerosol and lobar administration of a recombinant adenovirus to individuals with cystic fibrosis. II. Transfection efficiency in airway epithelium

A phase I clinical trial was conducted in which recombinant adenovirus containing the cystic fibrosis trans-membrane regulator (CFTR) (Ad2/CFTR) was administered by bronchoscopic instillation or aerosolization to the lungs of cystic fibrosis (CF) patients. In this paper, we evaluate the efficiency of Ad2/CFTR-mediated transduction of bronchial airway cells. The ability of an Ad2/CFTR vector to transduce airway cells was first evaluated in patients to whom the vector was administered by bronchoscopic instillation. Cells at the administration site were collected 2 days after treatment by bronchoscopic brushing. Ad2-specific CFTR DNA was detected in four of five individuals by PCR, and Ad2-specific CFTR RNA was detected in three of five individuals by RT-PCR. Ad2/CFTR-mediated transduction of airway epithelial cells was then determined in CF individuals receiving this vector by aerosol inhalation. Ad2-specific CFTR DNA was detected in 13 of 13 individuals 2 days after aerosolization, and in 3 of 5 individuals 7 days after aerosolization. Ad2-specific RNA was detected in 4 of 13 individuals on day 2, but was not detected in the 5 individuals tested on day 7. The percentage of airway epithelial cells containing nuclear-localized vector DNA was < or =2.4% as determined by fluorescence in situ hybridization (FISH). However, in some cases, a high percentage of nonepithelial mononuclear cells or squamous metaplastic epithelial cells was infected with the adenoviral vector. In conclusion, aerosol administration is a feasible means to distribute adenoviral vectors throughout the conducting airways, but improvements in adenovirus-mediated transduction of airway epithelial cells are necessary before gene therapy for CF will be effective.     

Airway-directed gene transfer of interleukin-10 using recombinant Sendai virus effectively prevents post-transplant fibrous airway obliteration in mice

Bronchiolitis obliterans (BO) after lung transplantation prevents a satisfactory prognosis, and recent studies suggested that interleukin-10 (IL-10) gene transfer to distant organs could inhibit BO in rodent models. Although delivery of the therapeutic gene to a local airway would be favored to minimize systemic effects, current limitations include lower gene transfer efficiency to airway epithelium. As recombinant Sendai virus (SeV) can produce dramatically efficient gene transfer to airway epithelium, we determined if SeV-mediated IL-10 gene transfer to the local airway would inhibit bronchial fibrous obliteration in murine tracheal allografts. Administration of cyclosporine A (CsA) significantly promoted not only recovery of the injured airway epithelium but also SeV-mediated IL-10 expression (CsA- versus CsA+ =228+/-78 versus 3627+/-1372 pg/graft with 5 x 10(7) pfu), thereby suggesting the requirement of epithelia for efficient gene transfer. Even at the highest expression, no significant leakage of IL-10 was evident in the systemic circulation, and the induction of interferon-gamma was completely diminished on day 7 by IL-10 gene transfer. As a result, luminal loss was significantly prevented in allografts treated with SeV-IL-10 (luminal opening, all control groups: 0% respectively, and SeV-IL-10 5 x 10(7) pfu: 25.7+/-10.5%), an effect that was enhanced by short-term CsA treatment (SeV-IL-10 5 x 10(7) pfu with CsA: 63.7+/-12.7%). We propose that SeV is a useful vector that can target airway epithelium to prevent BO avoiding putative systemic effect.     

Epithelial reticulon 4B (Nogo-B) is an endogenous regulator of Th2-driven lung inflammation

Nogo-B is a member of the reticulon family of proteins (RTN-4B) that is highly expressed in lung tissue; however, its function remains unknown. We show that mice with Th2-driven lung inflammation results in a loss of Nogo expression in airway epithelium and smooth muscle compared with nonallergic mice, a finding which is replicated in severe human asthma. Mice lacking Nogo-A/B (Nogo-KO) display an exaggerated asthma-like phenotype, and epithelial reconstitution of Nogo-B in transgenic mice blunts Th2-mediated lung inflammation. Microarray analysis of lungs from Nogo-KO mice reveals a marked reduction in palate lung and nasal clone (PLUNC) gene expression, and the levels of PLUNC are enhanced in epithelial Nogo-B transgenic mice. Finally, transgenic expression of PLUNC into Nogo-KO mice rescues the enhanced asthmatic-like responsiveness in these KO mice. These data identify Nogo-B as a novel protective gene expressed in lung epithelia, and its expression regulates the levels of the antibacterial antiinflammatory protein PLUNC.     

Enhancement of adenovirus-mediated gene transfer to the airways by DEAE dextran and sodium caprate in vivo

Gene transfer to the trachea and airways by adenoviral vectors is limited by the basolateral localization of viral receptors, resulting in relatively low levels of transduction. Modification of paracellular permeability by sodium caprate, which opens tight junctions, enhances gene transfer from the apical side of cultured human airway epithelial cells. Based on this observation we investigated whether Na-caprate could also increase gene transfer when applied to the luminal surface of the airway epithelia in vivo and compared these results with EGTA, which has previously been shown to enhance adenovirus transduction. Transgene expression in the trachea and upper airways was increased 25-fold by a 10-min pretreatment with 50 mM Na-caprate, corresponding to a 3-fold improvement over EGTA. In the more peripheral airways EGTA had no effect, whereas expression of β-gal was increased 3-fold by Na-caprate. When the adenovirus was complexed with DEAE dextran, transduction of the airway epithelia after Na-caprate pretreatment was increased 45-fold over virus alone. In conclusion, Na-caprate facilitates gene transfer to airway epithelia, particularly when adenovirus is complexed with DEAE dextran, and may in future be used in a clinical setting to enhance the efficiency of vectors for gene therapy of cystic fibrosis via airway delivery.