David R. Koehler

Protection of Cftr knockout mice from acute lung infection by a helper-dependent adenoviral vector expressing Cftr in airway epithelia

We developed a helper-dependent adenoviral vector for cystic fibrosis lung gene therapy. The vector expresses cystic fibrosis transmembrane conductance regulator (Cftr) using control elements from cytokeratin 18. The vector expressed properly localized CFTR in cultured cells and in the airway epithelia of mice. Cftr RNA and protein were present in whole lung and bronchioles, respectively, for 28 days after a vector dose. Acute inflammation was minimal to moderate. To test the therapeutic potential of the vector, we challenged mice with a clinical strain of Burkholderia cepacia complex (Bcc). Cftr knockout mice (but not Cftr+/+ littermates) challenged with Bcc developed severe lung histopathology and had high lung bacteria counts. Cftr knockout mice receiving gene therapy 7 days before Bcc challenge had less severe histopathology, and the number of lung bacteria was reduced to the level seen in Cftr+/+ littermates. These data suggest that gene therapy could benefit cystic fibrosis patients by reducing susceptibility to opportunistic pathogens.

Reduced inflammation and improved airway expression using helper-dependent adenoviral vectors with a K18 promoter.

Efforts have been made to deliver transgenes to the airway epithelia of laboratory animals and humans to develop gene therapy for cystic fibrosis. These investigations have been disappointing due to combinations of transient and low-level gene expression, acute toxicity, and inflammation. We have developed new helper-dependent adenoviral vectors to deliver an epithelial cell-specific keratin 18 expression cassette driving the beta-galactosidase (beta-gal) or human alpha-fetoprotein (AFP) reporter genes. Following intranasal administration to mice, we found that the reporter genes were widely expressed in airway epithelial and submucosal cells, and secreted human AFP was also detectable in serum. In contrast to a first-generation adenoviral vector, inflammation was negligible at doses providing efficient transduction, and expression lasted longer than typically reported-up to 28 days with beta-gal and up to 15 weeks with human AFP. These results suggest that delivery to the airway of helper-dependent adenoviral vectors utilizing a tissue-specific promoter could be a significant advance in the development of gene therapy for cystic fibrosis.

A novel developmentally regulated gene in lung mesenchyme: homology to a tumor-derived trypsin inhibitor.

We used differential display-PCR (DD-PCR) to identify glucocorticoid-inducible genes that regulate lung development in late gestation. DD-PCR, a method to screen for differentially expressed genes, is based on a comparison of mRNAs isolated from a subset of two or more cell populations by analysis of RT-PCR products on DNA-sequencing gels. We isolated cDNA probes representing mRNAs expressed in primary cultures of rat lung fibroblasts, but not in epithelial cells, on fetal day 20. A day 20 glucocorticoid-treated fibroblast cDNA library was screened with a single probe to isolate the 3.1-kb cDNA late-gestation lung 1( LGL1; GenBank accession no. AF109674 ) encoding a deduced polypeptide of 188 amino acids. Northern analysis confirmed that LGL1is expressed in human, rat, and mouse fetal lungs, induced by glucocorticoid, developmentally regulated in fibroblasts but not detectable in epithelium. In situ hybridization confirmed LGL1 expression in the mesenchyme, but not in the epithelium, of fetal rat lung, kidney, and gut. The predicted LGL1 gene product (lgl1) showed 81% homology to P25TI, a polypeptide trypsin inhibitor recently identified in human glioblastoma and neuroblastoma cells but not detected in normal human tissues. Both lgl1 and P25TI belong to the CRISP family of cysteine-rich extracellular proteins. Trypsin is produced by both normal bronchial epithelial and lung adenocarcinoma cells. Although additional studies will be necessary to clearly establish a functional role for lgl1, we propose that lgl1 has a role in normal lung development that is likely to be via regulation of extracellular matrix degradation.

Aerosol delivery of an enhanced helper-dependent adenovirus formulation to rabbit lung using an intratracheal catheter

Poor transduction of the ciliated airway epithelium and inefficient airway delivery of viral vectors are common difficulties encountered in lung gene therapy trials with large animals and humans.

Regulation of epithelium-specific Ets-like factors ESE-1 and ESE-3 in airway epithelial cells: potential roles in airway inflammation.

Airway inflammation is the hallmark of many respiratory disorders, such as asthma and cystic fibrosis. Changes in airway gene expression triggered by inflammation play a key role in the pathogenesis of these diseases. Genetic linkage studies suggest that ESE-2 and ESE-3, which encode epithelium-specific Ets-domain-containing transcription factors, are candidate asthma susceptibility genes. We report here that the expression of another member of the Ets family transcription factors ESE-1, as well as ESE-3, is upregulated by the inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in bronchial epithelial cell lines. Treatment of these cells with IL-1β and TNF-α resulted in a dramatic increase in mRNA expression for both ESE-1 and ESE-3. We demonstrate that the induced expression is mediated by activation of the transcription factor NF-κB. We have characterized the ESE-1 and ESE-3 promoters and have identified the NF-κB binding sequences that are required for the cytokine-induced expression. In addition, we also demonstrate that ESE-1 upregulates ESE-3 expression and downregulates its own induction by cytokines. Finally, we have shown that in Elf3 (homologous to human ESE-1) knockout mice, the expression of the inflammatory cytokine interleukin-6 (IL-6) is downregulated. Our findings suggest that ESE-1 and ESE-3 play an important role in airway inflammation.

Readministration of helper-dependent adenovirus to mouse lung.

Adenovirus vectors (Ad) are widely used in gene therapy studies, including those aimed at treating cystic fibrosis lung disease. Various approaches have been investigated to blunt the host immune response to Ad, including development of helper-dependent (HD) Ad. The host cytotoxic T-cell response to HD-Ad is generally lower than to earlier-generation Ad. However, antibodies are formed which could inhibit the efficacy of HD-Ad readministration. In this first study of HD-Ad readministration to the lung, we found that a second administration of HD-Ad to mice was possible with minimal loss of transgene expression. In contrast, when first-generation (FG) Ad was administered initially, followed by HD-Ad or FG-Ad, transgene expression was reduced. Significantly lower concentrations of antibodies against Ad were found in lung lavage fluid and serum from mice that received two doses of HD-Ad (when the initial HD-Ad lacked a transgene), compared to mice that received FG-Ad followed by HD-Ad. These data suggest that readministration of HD-Ad for lung gene therapy may be feasible.

Down-regulation of IL-8 expression in human airway epithelial cells through helper-dependent adenoviral-mediated RNA interference

ABSTRACTInterleukin (IL)-8 is a potent neutrophil chemotactic factor and a crucial mediator in neutrophil-dependent inflammation. Various cell types produce IL-8, either in response to external stimuli such as cytokines or bacterial infection, or after malignant transformation. Anti-IL-8 strategies have been considered for anti-inflammatory therapy. In this paper we demonstrate that the RNA interference technique can be used to efficiently down-regulate IL-8 protein expression in airway epithelial cells. We used a helper-dependent adenoviral vector to express a small hairpin (sh)RNA targeting human IL-8 in cultured airway epithelial cells (IB3-1, Cftr−/−; C38, Cftr-corrected) stimulated with TNF-α, IL-1β or heat-inactivated Burkholderia cenocepacia. Stimulated IL-8 expression in IB3-1 and C38 cells was significantly reduced by shRNA expression. The shRNA targeting IL-8 had no effect on the activation of NF-κB, or on the protein levels of IκB or IL-6, suggesting that this anti-IL-8 strategy was highly specific, and therefore may offer potential for the treatment of inflammatory diseases.

A Human Epithelium-Specific Vector Optimized in Rat Pneumocytes for Lung Gene Therapy

Gene therapy vectors based on mammalian promoters offer the potential for increased cell specificity and may be less susceptible than viral promoters to transcription attenuation by host cytokines. The human cytokeratin 18 (K18) gene is naturally expressed in the lung epithelia, a target site for gene therapies to treat certain genetic pediatric lung diseases. Our original vector based on the promoter and 5′ control elements of K18 offered excellent epithelial cell specificity but relatively low expression levels compared with viral promoters. In the present study, we found that adding a stronger SV40 poly(A) signal boosted primary rat lung epithelial cell expression but greatly reduced cell specificity. Addition of a 3′ portion of the K18 gene to our vector as a 3′ untranslated region (UTR) improved epithelial cell-specific expression by reducing expression in lung fibroblasts. The effect of the 3′ UTR was not related to gross differences in cell-specific splicing. A deletion variant of this UTR further increased lung epithelial cell expression while retaining some cell specificity. These data illustrate the possibilities for using 3′ UTR to regulate cell-specific transgene expression. Our improved K18 vector should prove useful for pediatric lung gene therapy applications.

363. Gene Delivery to Human Sweat Glands: A Model for Cystic Fibrosis Gene Therapy

Cystic fibrosis (CF) is considered a disease that could be treated by gene therapy, yet the results from past clinical trials showed only transient transgene expression. Gene therapy vectors are mostly studied in cultured cells, rodent models or non-human primates, but it is difficult to test them in human system prior to clinical studies. In this study, we investigated the possibility of using human sweat glands as a model for testing CF gene therapy vectors. In order to deliver genes to human sweat glands ex vivo, we explored various gene delivery methods using a helper-dependent adenovirus vector containing cytokeratin 18 regulatory elements. Gene delivery to sweat glands in skin organ culture by topical application, intra-dermal injection or submerged culture was not effective. We were able to enhance transduction efficiency by isolating and pre-treating sweat glands with dispase, which indicates that the basement membrane is a critical barrier to gene delivery by adenoviral vectors. Using this approach, we showed that the Cftr gene could be efficiently delivered to and expressed by the epithelial cells of sweat glands with our helper dependent adenoviral vector. Based on our study we propose that sweat glands can be used as an alternative model to study functional efficacy of CF gene therapy in humans.

110. Helper-Dependent Adenovirus with the Cytokeratin-18 Promoter Produces Greater Transepithelial Chloride Current and More Persistent Transgene Expression Than the CMV Promoter in Differentiated Human Airway Epithelia

Gene transfer of the wild-type CFTR cDNA to airway epithelia could rescue the electrolyte transport defect and represent an attractive strategy for treating the genetic disease cystic fibrosis (CF). Numerous gene transfer vectors have expressed CFTR under control of the cytomegalovirus (CMV) enhancer/promoter because it generates robust expression in many cell types. However, this promoter could drive expression in cell types that do not normally express CFTR and may yield limited persistence. An alternative enhancer/promoter is from the cytoskeletal protein cytokeratin-18 (K18); cytokeratin-18 is constitutively expressed in differentiated epithelia and has a tissue distribution similar to CFTR. To test the K18 promoter, we used helper-dependent adenoviral vectors and compared transgene expression driven by the K18 and CMV promoters. We delivered them to well-differentiated primary cultures of CF airway epithelia grown at the air-liquid interface. We applied vector to the basolateral surface to facilitate infection through binding of the adenovirus receptor CAR. With a β-galactosidase transgene, the CMV promoter generated much greater β-galactosidase activity and total protein levels than the K18 promoter. However, the opposite pattern emerged when the two promoters drove CFTR expression; the K18 promoter generated greater transepithelial chloride current than the CMV promoter. These results suggested that the two promoters might drive expression in different cell types. We found that the CMV promoter generated the most intense X-Gal staining in basal cells. In contrast, the K18 promoter generated diffuse staining in columnar epithelial cells. These results suggest that the K18 promoter produced relatively greater CFTR expression in columnar epithelial cells that touch the apical surface and are responsible for transepithelial chloride transport, whereas the CMV promoter generated greater expression in basal cells. We also compared the persistence of transgene expression. Over the course of 12 weeks, transgene expression driven by the K18 promoter remained relatively constant, while transgene expression from the CMV promoter fell rapidly. These data emphasize the importance of appropriate regulatory elements in developing gene transfer vectors for CF airway epithelia and suggest that the K18 promoter may have advantages over the CMV promoter.