Therapy For Cystic Fibrosis Lung Disease Research Articles

IL-17A from innate and adaptive lymphocytes contributes to inflammation and damage in cystic fibrosis lung disease.

Elevated levels of interleukin (IL)-17A were detected in the airways of patients with cystic fibrosis (CF), but its cellular sources and role in the pathogenesis of CF lung disease remain poorly understood. The aim of this study was to determine the sources of IL-17A and its role in airway inflammation and lung damage in CF. We performed flow cytometry to identify IL-17A-producing cells in lungs and peripheral blood from CF patients and β-epithelial Na+ channel transgenic (Scnn1b-Tg) mice with CF-like lung disease, and determined the effects of genetic deletion of Il17a and Rag1 on the pulmonary phenotype of Scnn1b-Tg mice. T-helper 17 cells, CD3+CD8+ T-cells, γδ T-cells, invariant natural killer T-cells and innate lymphoid cells contribute to IL-17A secretion in lung tissue, lymph nodes and peripheral blood of patients with CF. Scnn1b-Tg mice displayed increased pulmonary expression of Il17a and elevated IL-17A-producing innate and adaptive lymphocytes with a major contribution by γδ T-cells. Lack of IL-17A, but not the recombination activating protein RAG1, reduced neutrophilic airway inflammation in Scnn1b-Tg mice. Genetic deletion of Il17a or Rag1 had no effect on mucus obstruction, but reduced structural lung damage and revealed an IL-17A-dependent macrophage activation in Scnn1b-Tg mice. We identify innate and adaptive sources of IL-17A in CF lung disease. Our data demonstrate that IL-17A contributes to airway neutrophilia, macrophage activation and structural lung damage in CF-like lung disease in mice. These results suggest IL-17A as a novel target for anti-inflammatory therapy of CF lung disease.

Viral Vectors, Animal Models, and Cellular Targets for Gene Therapy of Cystic Fibrosis Lung Disease.

After more than two decades since clinical trials tested the first use of recombinant adeno-associated virus (rAAV) to treat cystic fibrosis (CF) lung disease, gene therapy for this disorder has undergone a tremendous resurgence. Fueling this enthusiasm has been an enhanced understanding of rAAV transduction biology and cellular processes that limit transduction of airway epithelia, the development of new rAAV serotypes and other vector systems with high-level tropism for airway epithelial cells, an improved understanding of CF lung pathogenesis and the cellular targets for gene therapy, and the development of new animal models that reproduce the human CF disease phenotype. These advances have created a preclinical path for both assessing the efficacy of gene therapies in the CF lung and interrogating the target cell types in the lung required for complementation of the CF disease state. Lessons learned from early gene therapy attempts with rAAV in the CF lung have guided thinking for the testing of next-generation vector systems. Although unknown questions still remain regarding the cellular targets in the lung that are required or sufficient to complement CF lung disease, the field is now well positioned to tackle these challenges. This review will highlight the role that next-generation CF animal models are playing in the preclinical development of gene therapies for CF lung disease and the knowledge gaps in disease pathophysiology that these models are attempting to fill.

Establishment of a High-Yield Recombinant Adeno-Associated Virus/Human Bocavirus Vector Production System Independent of Bocavirus Nonstructural Proteins.

The genome of recombinant adeno-associated virus 2 (rAAV2) remains a promising candidate for gene therapy for cystic fibrosis (CF) lung disease, but due to limitations in the packaging capacity and the tropism of this virus with respect to the airways, strategies have evolved for packaging an rAAV2 genome (up to 5.8 kb) into the capsid of human bocavirus 1 (HBoV1) to produce a chimeric rAAV2/HBoV1 vector. Although a replication-incompetent HBoV1 genome has been established as a trans helper for capsid complementation, this system remains suboptimal with respect to virion yield. Here, a streamlined production system is described based on knowledge of the involvement of HBoV1 nonstructural (NS) proteins NS1, NS2, NS3, NS4, and NP1 in the process of virion production. The analyses reveal that NS1 and NS2 negatively impact virion production, NP1 is required to prevent premature termination of transcription of the cap mRNA from the native genome, and silent mutations within the polyadenylation sites of the cap coding sequence can eliminate this requirement for NP1. It is further shown that preventing the expression of all NS proteins significantly increases virion yield. Whereas the expression of capsid proteins VP1, VP2, and VP3 from a codon-optimized cap mRNA was highly efficient, optimal virion assembly, and thus potency, required enhanced VP1 expression, entailing a separate VP1 expression cassette. The final NS protein-free production system uses three-plasmid co-transfection of HEK293 cells, with one trans helper plasmid encoding VP1 and the AAV2 Rep proteins, and another encoding VP2-3 and components from adenovirus. This system yielded >16-fold more virions than the prototypic system, without reducing transduction potency. This increase in virion production is expected to facilitate greatly both research on the biology of rAAV2/HBoV1 and preclinical studies testing the effectiveness of this vector for gene therapy of CF lung disease in large animal models.

574. Sustaining Helper-Dependent Adenoviral Vector-Mediated Human CFTR Expression in Mouse Airways Through Transient Immunosuppression

Cystic Fibrosis (CF) is a common life-threatening disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an epithelial chloride channel. Gene therapy for CF lung disease has been tested extensively. However, sustained expression of the CFTR gene is a major challenge to gene delivery with either viral or no viral vectors. One strategy to improve the persistent CFTR expression is to modulate host immune responses by transient immunosuppression.We showed previously that cyclophosphamide significantly enhanced human CFTR expression in mice received HD-Ad vector readministration, through inhibition of host immune reactions. In this study, we examined cyclophosphamide, dexmethoson, and combination of cyclosporine, methylprednsolone and azathioprine, for their effects on long term expression of the human CFTR delivered with helper-dependent adenoviral (HD-Ad) vectors to mouse airways. Four group mice were nasally delivered with an HD-Ad-K18-CFTR vector at 1.5×10E10 particles per mouse. Three groups were treated with immunesuppresants and one group without any treatment was used as a control. The immunosuppressants were administrated twice, at 6 hours before and 48 hours after nasal delivery of the HD-Ad-CFTR vector. Expression of the CFTR gene was assessed 4 months after vector administration. We found that cyclophosphamide has significantly improved the sustained expression of the CFTR gene (2.5 times higher) compared to the no treatment group as determined by real-time qPCR. It alsogreatly reduced the levels of anti-adenoviral and neutralizing antibodies in both BALF and serum. However, cyclophosphamide did not show major effects on the innate immune reaction to HD-Ad vectors, such as NK cell infiltration and activation, 4 days after vector delivery. Dexmethesone also greatly reduced neutralizing antibodies production, but not affected transgene expression. A combination of cyclosporine, methylprednsolone and azathioprine which is used to suppress immune reactions in lung transplantation, also did not affect CFTR gene expression, nor immune reactions following HD-Ad-CFTR vector delivery. These data demonstrate that transient administration of cyclophosphamide can significantly improve sustained HD-Ad-mediated transgene expression in mice and it is more effective than dexmethsone or the combination of cyclosporine, methylprednsolone and azathioprine.

The triterpenoid CDDO limits inflammation in preclinical models of cystic fibrosis lung disease

Excessive inflammation in cystic fibrosis (CF) lung disease is a contributor to progressive pulmonary decline. Effective and well-tolerated anti-inflammatory therapy may preserve lung function, thereby improving quality and length of life. In this paper, we assess the anti-inflammatory effects of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) in preclinical models of CF airway inflammation. In our experiments, mice carrying the R117H Cftr mutation have significantly reduced airway inflammatory responses to both LPS and flagellin when treated with CDDO before inflammatory challenge. Anti-inflammatory effects observed include reduced airway neutrophilia, reduced concentrations of proinflammatory cytokines and chemokines, and reduced weight loss. Our findings with the synthetic triterpenoids in multiple cell culture models of CF human airway epithelia agree with effects previously described in other disease models (e.g., neoplastic cells). These include the ability to reduce NF-kappaB activation while increasing nuclear factor erythroid-related factor 2 (Nrf2) activity. As these two signaling pathways appear to be pivotal in regulating the net inflammatory response in the CF airway, these compounds are a promising potential anti-inflammatory therapy for CF lung disease.

Non-viral vectors in cystic fibrosis gene therapy: recent developments and future prospects

Gene therapy has been proposed for a wide range of human diseases but few have received the level of attention over such a prolonged period as cystic fibrosis (CF) with over 20 clinical studies undertaken. Following a 10-year interval, clinical trials of an aerosolisable non-viral gene transfer agent have recently been initiated by researchers in the United Kingdom. Here we review the rationale and requirements for effective gene therapy for CF lung disease. The previous non-viral gene therapy trials are discussed and the prospects for the current leading non-viral formulations for CF gene therapy are considered. Factors affecting the selection and design of the plasmid DNA molecule, likely to be of central importance to clinical efficacy, are reviewed and we describe the potential merits of the formulation that has been selected for the forthcoming UK trials.

Cystic fibrosis: recent advances in lung-directed therapies

Cystic fibrosis (CF) lung disease is characterized by progressively worsening pulmonary infections, airway obstruction and ultimately early death. Current pharmacotherapeutic treatments can only alleviate the secondary symptoms associated with lung disease. Alternative therapeutic strategies are now being developed to target the CF gene defect at the molecular level. Preclinical and clinical trials are currently underway and early results strongly suggest that these strategies can be translated into an effective curative therapy for CF lung disease.

Physical and Biological Barriers to Viral Vector-mediated Delivery of Genes to the Airway Epithelium

A gene therapy for cystic fibrosis (CF) lung disease by intralumenal delivery of therapeutic transgenes into the lung is a logical treatment strategy if efficient gene transfer can be achieved without detrimental effects to the patient. Indeed, pioneering work in the late 1980s showed that genetically engineered viruses could deliver the CF corrective transgene to cultured cells from patients with CF. However, after many attempts to deliver the corrective gene to the lungs of patients with CF in vivo and with the luxury of 20/20 hindsight, it is realized that although logical, the strategy to accomplish this task did not appreciate the evolution of the lung to resist invasion by pathogens such as viruses. It is now apparent that several levels of barriers exist that restrict exogenous gene delivery to the airway epithelium by commonly used viral vectors. Components of the innate and cell-mediated immune system collectively limit both the access to and duration of gene transfer vectors to the airway epithelium. Alternative viral vectors that have evolved to circumvent these barriers will require further development if gene transfer is ever to be considered a therapy for CF lung disease.

480. Magnetofection To Enhance Transfection Efficiency of Large Genomic Context Vectors In Vitro

Cystic fibrosis (CF) is the most common recessive lethal inherited disorder among Caucasian populations. The genetic basis of cystic fibrosis was resolved in 1989 when the CFTR (cystic fibrosis transmembrane conductance regulator) gene was cloned. Until recently, few CF patients survived childhood and other than transplantation, there is no effective therapy for CF lung disease. The realisation of gene therapy represents the best hope for these patients.

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.

Opportunities for the Use of Aerosolized α1-Antitrypsin for the Treatment of Cystic Fibrosis

Cystic fibrosis (CF), the most common lethal genetic disease affecting the white population, owes its morbidity and mortality primarily to the devastating effects of chronic inflammation and infection within the pulmonary airways. It has become increasingly recognized that the host's response to Pseudomonas species and Staphylococcus aureus infection plays a paramount role in CF lung destruction and eventual development of respiratory insufficiency. A massive pulmonary influx of neutrophils, and accompanying excessive levels of neutrophil elastase (NE), can be detected in the bronchoalveolar fluid of even very young children with CF. The excess of NE adversely affects the CF airways by enhancing mucus secretion, directly injuring airway tissues, exacerbating the inflammatory process by attracting more neutrophils, and derailing opsonization and elimination of bacterial pathogens, particularly Pseudomonas aeruginosa. Neutralization of excess NE by delivering supplemental alpha 1-antitrypsin to the airways via aerosolization represents an exciting new potential therapy for CF lung disease.

Tyloxapol inhibits NF-kappa B and cytokine release, scavenges HOCI, and reduces viscosity of cystic fibrosis sputum.

Cystic fibrosis (CF) patients develop progressive cytokine-mediated inflammatory lung disease, with abundant production of thick, tenacious, protease- and oxidant-rich purulent airway secretions that are difficult to clear even with physiotherapy. In the search for a potential treatment, we have tested tyloxapol, an alkylaryl polyether alcohol polymer detergent previously used as a mucolytic agent in adult chronic bronchitis. Tyloxapol inhibits activation of the transcription factor nuclear factor-kappa B (NK-kappa B), reduces resting secretion of the cytokine interleukin-8 (IL-8) in cultured human monocytes, and inhibits lipopolysaccharide (LPS)-stimulated release of tumor necrosis factor-alpha (TNF-alpha), IL-1 beta, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), and the eiconsanoids thromboxane A2 and leukotriene B4 (LTB4). We have previously shown that tyloxapol is a potent antioxidant for hydroxyl radicals ( OH). Tyloxapol (0.05 to 0.1% wt/vol) effectively scavenges the oxidant hypochlorous acid (HOCl; 1 to 7.5 mM) in vitro, and protects from HOCl-mediated lung injury in rats. Tyloxapol also reduces the viscosity of CF sputum (from 463 +/- 133 to 128 +/- 52 centipoise). We conclude that tyloxapol is potentially useful as a new antiinflammatory therapy for CF lung disease, and could possibly promote clearance of secretions in the CF airway.

Inefficient gene transfer by adenovirus vector to cystic fibrosis airway epithelia of mice and humans

The success of adenoviral vectors for gene therapy of lung disease in cystic fibrosis (CF) depends on efficient transfer of the complementary DNA encoding the correct version of the cystic fibrosis transmembrane regulator (CFTR) to the affected columnar epithelial cells lining the airways of the lung. Pre-clinical studies in vitro suggest that low doses of adenovirus vectors carrying this CFTR cDNA can correct defective Cl- transport in cultured human CF airway epithelia. Here we use mice carrying the disrupted CF gene to test the efficacy of this transfer system in vivo. We find that even repeated high doses can only partially (50%) correct the CF defect in Cl- transport in vivo and do not correct the Na+ transport defect at all. We investigated this discrepancy between the in vivo and in vitro transfer efficiency using CF mouse and human samples, and found that it reflects a difference in the susceptibility to adenovirus-5 transduction of the epithelial cell types dosed in vivo (columnar) and in vitro (basal-cell-like). These studies indicate that more efficient adenoviral gene-transfer vectors and/or refinement of dosing strategies are needed for therapy of CF lung disease.