Nebulisation of receptor-targeted nanocomplexes for gene delivery to the airway epithelium

Background

Gene therapy mediated by synthetic vectors may provide opportunities for new treatments for cystic fibrosis (CF) via aerosolisation. Vectors for CF must transfect the airway epithelium efficiently and not cause inflammation so they are suitable for repeated dosing. The inhaled aerosol should be deposited in the airways since the cystic fibrosis transmembrane conductance regulator gene (CFTR) is expressed predominantly in the epithelium of the submucosal glands and in the surface airway epithelium. The aim of this project was to develop an optimised aerosol delivery approach applicable to treatment of CF lung disease by gene therapy.

Methodology

The vector suspension investigated in this study comprises receptor-targeting peptides, cationic liposomes and plasmid DNA that self-assemble by electrostatic interactions to form a receptor-targeted nanocomplex (RTN) of approximately 150 nm with a cationic surface charge of +50 mV. The aerodynamic properties of aerosolised nanocomplexes produced with three different nebulisers were compared by determining aerosol deposition in the different stages of a Next Generation Pharmaceutical Impactor (NGI). We also investigated the yield of intact plasmid DNA by agarose gel electrophoresis and densitometry, and transfection efficacies in vitro and in vivo.

Results

RTNs nebulised with the AeroEclipse II BAN were the most effective, compared to other nebulisers tested, for gene delivery both in vitro and in vivo. The biophysical properties of the nanocomplexes were unchanged after nebulisation while the deposition of RTNs suggested a range of aerosol aerodynamic sizes between 5.5 µm–1.4 µm cut off (NGI stages 3–6) compatible with deposition in the central and lower airways.

Conclusions

RTNs showed their ability at delivering genes via nebulisation, thus suggesting their potential applications for therapeutic interventions of cystic fibrosis and other respiratory disorders.     

Disrupted tight junctions in the small intestine of cystic fibrosis mice

The tight junction (TJ) is the major determinant of paracellular permeability, which in the gut protects the body from entry of harmful substances such as microbial components. In cystic fibrosis (CF), there is increased permeability of the small intestine both in humans and in CF mice. To gain insight into the mechanisms of increased intestinal permeability in CF, I analyze the composition of the TJ in a cystic fibrosis transmembrane conductance regulator (Cftr) knockout mouse model. Significant changes in TJ gene expression in the CF intestine were found for Cldn1, Cldn7, Cldn8 and Pmp22, which were expressed at lower levels and Cldn2 that was expressed at a higher level. Protein levels of claudin-2 were increased in the CF intestine as compared to wild-type, while other TJ proteins were not significantly different. In the villus epithelium of the CF intestine, all TJ components analyzed were mislocalized to the basal cytoplasm and showed varying degrees of loss from the TJ and apico-lateral surfaces. The pore-forming claudin-2 in the CF intestine showed more intense staining but was correctly localized to the TJ, principally in the crypts that are enlarged in CF. The cytokine TNFα, known to affect TJ, was elevated to 160 % of wild-type in the CF intestine. In summary, there is a dramatic redistribution of claudin proteins from the TJ/lateral membrane to the basal cytoplasm of the villus epithelium in the CF intestine. These changes in TJ protein localization in CF are likely to be involved in the increased permeability of the CF small intestine to macromolecules and TNFα may be a causative factor.     

Draft Genome Sequence Determination for Cystic Fibrosis and Chronic Granulomatous Disease Burkholderia multivorans Isolates

Burkholderia multivorans is a Gram-negative bacterium and a member of the Burkholderia cepacia complex, which is frequently associated with respiratory infections in people with cystic fibrosis (CF) and chronic granulomatous disease (CGD). We are reporting the genome sequences of 4 B. multivorans strains, 2 from CF patients and 2 from CGD patients.     

Pseudotyped AAV Vector-Mediated Gene Transfer in a Human Fetal Trachea Xenograft Model: Implications for In Utero Gene Therapy for Cystic Fibrosis

Background

Lung disease including airway infection and inflammation currently causes the majority of morbidities and mortalities associated with cystic fibrosis (CF), making the airway epithelium and the submucosal glands (SMG) novel target cells for gene therapy in CF. These target cells are relatively inaccessible to postnatal gene transfer limiting the success of gene therapy. Our previous work in a human-fetal trachea xenograft model suggests the potential benefit for treating CF in utero. In this study, we aim to validate adeno-associated virus serotype 2 (AAV2) gene transfer in a human fetal trachea xenograft model and to compare transduction efficiencies of pseudotyping AAV2 vectors in fetal xenografts and postnatal xenograft controls.

Methodology/Principal Findings

Human fetal trachea or postnatal bronchus controls were xenografted onto immunocompromised SCID mice for a four-week engraftment period. After injection of AAV2/2, 2/1, 2/5, 2/7 or 2/8 with a LacZ reporter into both types of xenografts, we analyzed for transgene expression in the respiratory epithelium and SMGs. At 1 month, transduction by AAV2/2 and AAV2/8 in respiratory epithelium and SMG cells was significantly greater than that of AAV2/1, 2/5, and 2/7 in xenograft tracheas. Efficiency in SMG transduction was significantly greater in AAV2/8 than AAV2/2. At 3 months, AAV2/2 and AAV2/8 transgene expression was >99% of respiratory epithelium and SMG. At 1 month, transduction efficiency of AAV2/2 and AAV2/8 was significantly less in adult postnatal bronchial xenografts than in fetal tracheal xenografts.

Conclusions/Significance

Based on the effectiveness of AAV vectors in SMG transduction, our findings suggest the potential utility of pseudotyped AAV vectors for treatment of cystic fibrosis. The human fetal trachea xenograft model may serve as an effective tool for further development of fetal gene therapy strategies for the in utero treatment of cystic fibrosis.     

Specific antibodies to recombinant allergens of Aspergillus fumigatus in cystic fibrosis patients with ABPA

Background

Aspergillus fumigatus, a widely distributed fungus, has been implicated in causing life threatening infections as well as severe asthma and allergic diseases in man. Allergic affliction like allergic bronchopulmonary aspergillosis (ABPA) is a disabling lung disease frequently seen in patients with asthma and cystic fibrosis. Immunodiagnosis of the former is comparatively easier due to the availability of purified antigens and sensitive methods. However, this is not true with cystic fibrosis patients where the prevalence of ABPA is fairly high and the morbidity and mortality are significant.

Methods

In the present study, we have evaluated purified recombinant allergens from A. fumigatus, namely Asp f 1, f 2, f 3, f 4, and f 6 using ELISA and a semi-automated method (ImmunoCAP). We studied 17 patients each from cystic fibrosis with ABPA, and cystic fibrosis with asthma, 22 cystic fibrosis with no ABPA or asthma, and 11 age matched controls.

Results

The results indicate that no antigen, antibody or method is capable of differentiating cystic fibrosis (CF) with ABPA from other CF patients, although some allergens showed strong reaction or showed more prevalence among the patients studied.

Conclusion

When results of several allergens such as Asp f 1, f 2, f 3, f 4, and f 6 in their binding to IgA, IgG, and IgE antibodies were analyzed, a more strong discrimination of CF patients with ABPA was possible from the other groups studied.     

Outwardly rectifying chloride channels and CF: A divorce and remarriage

Outwardly rectifying Cl^– channels were originally thought to be the central element in cystic fibrosis. The role of these channels in CF was questioned to such an extent that doubts were rasied about the validity of the original experiments. Recent data reestablishes a role for outwardly rectifying Cl^– channels (ORCC) in CF and suggests that the protein encoded by the CF gene, the cystic fibrosis transmembrane regulator (CFTR), can effect the regulation of more than one channel in the airway. This minireview deals with the rise, fall, and resurrection of the role of outwardly rectifying Cl^– channels in CF.     

Taking stock of gene therapy for cystic fibrosis

The identification of the cystic fibrosis (CF) gene opened the way for gene therapy. In the ten years since then, proof of principle in vitro and then in animal models in vivo has been followed by numerous clinical studies using both viral and non-viral vectors to transfer normal copies of the gene to the lungs and noses of CF patients. A wealth of data have emerged from these studies, reflecting enormous progress and also helping to focus and define key difficulties that remain unresolved. Gene therapy for CF remains the most promising possibility for curative rather than symptomatic therapy.     

Culture-Independent Identification of Nontuberculous Mycobacteria in Cystic Fibrosis Respiratory Samples

Respiratory tract infections with nontuberculous mycobacteria (NTM) are increasing in prevalence and are a significant cause of lung function decline in individuals with cystic fibrosis (CF). NTM have been detected in culture-independent analyses of CF airway microbiota at lower rates than would be expected based on published prevalence data, likely due to poor lysing of the NTM cell wall during DNA extraction. We compared a standard bacterial lysis protocol with a modified method by measuring NTM DNA extraction by qPCR and NTM detection with bacterial 16S rRNA gene sequencing. The modified method improved NTM DNA recovery from spiked CF sputum samples by a mean of 0.53 log₁₀ copies/mL for M. abscessus complex and by a mean of 0.43 log₁₀ copies/mL for M. avium complex as measured by qPCR targeting the atpE gene. The modified method also improved DNA sequence based NTM detection in NTM culture-positive CF sputum and bronchoalveolar lavage samples; however, both qPCR and 16S rRNA gene sequencing remained less sensitive than culture for NTM detection. We highlight the limitations of culture-independent identification of NTM from CF respiratory samples, and illustrate how alterations in the bacterial lysis and DNA extraction process can be employed to improve NTM detection with both qPCR and 16S rRNA gene sequencing.     

Allele frequency for Cystic fibrosis in Indians vis-a/-vis global populations

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene. This gene encodes a protein involved in epithelial anion channel. Cystic fibrosis is the most common life-limiting genetic disorder in Caucasians; it also affects other ethnic groups like the Blacks and the Native Americans. Cystic fibrosis is considered to be rare among individuals from the Indian subcontinent. We analyzed a total of 29 world׳s populations for cystic fibrosis on the basis of gene frequency and heterozygosity. Among 29 countries Switzerland revealed the highest gene frequency and heterozygosity for CF (0.022, 0.043) whereas Japan recorded the lowest values (0.002, 0.004) followed by India (0.004, 0.008). Our analysis suggests that the prevalence of cystic fibrosis is very low in India.     

Optical Coherence Tomography Identifies Lower Labial Salivary Gland Surface Density in Cystic Fibrosis

The labial minor salivary glands (LSGs) are easily accessible mucus-secreting structures of the alimentary tract that may provide new information on the basis of gastrointestinal complications of cystic fibrosis (CF). It was shown that they are destructed in the course of cystic fibrosis. We employed wide-field, micrometer resolution in vivo optical coherence tomography to assess the surface density of LSGs in 18 patients with CF and 18 healthy subjects. The median LSGs’ surface densities in CF patients, and in the control group were 4.32 glands/cm² and 6.58 glands/cm², respectively (p = 0.006; Mann-Whitney U test). A lower LSG surface density is a previously unrecognized CF-related pathology of the alimentary tract.     

Elevated Paracellular Glucose Flux across Cystic Fibrosis Airway Epithelial Monolayers Is an Important Factor for Pseudomonas aeruginosa Growth

People with cystic fibrosis (CF) who develop related diabetes (CFRD) have accelerated pulmonary decline, increased infection with antibiotic-resistant Pseudomonas aeruginosa and increased pulmonary exacerbations. We have previously shown that glucose concentrations are elevated in airway surface liquid (ASL) of people with CF, particularly in those with CFRD. We therefore explored the hypotheses that glucose homeostasis is altered in CF airway epithelia and that elevation of glucose flux into ASL drives increased bacterial growth, with an effect over and above other cystic fibrosis transmembrane conductance regulator (CFTR)-related ASL abnormalities. The aim of this study was to compare the mechanisms governing airway glucose homeostasis in CF and non-CF primary human bronchial epithelial (HBE) monolayers, under normal conditions and in the presence of Ps. aeruginosa filtrate. HBE-bacterial co-cultures were performed in the presence of 5 mM or 15 mM basolateral glucose to investigate how changes in blood glucose, such as those seen in CFRD, affects luminal Ps. aeruginosa growth. Calu-3 cell monolayers were used to evaluate the potential importance of glucose on Ps. aeruginosa growth, in comparison to other hallmarks of the CF ASL, namely mucus hyperviscosity and impaired CFTR-dependent fluid secretions. We show that elevation of basolateral glucose promotes the apical growth of Ps. aeruginosa on CF airway epithelial monolayers more than non-CF monolayers. Ps. aeruginosa secretions elicited more glucose flux across CF airway epithelial monolayers compared to non-CF monolayers which we propose increases glucose availability in ASL for bacterial growth. In addition, elevating basolateral glucose increased Ps. aeruginosa growth over and above any CFTR-dependent effects and the presence or absence of mucus in Calu-3 airway epithelia-bacteria co-cultures. Together these studies highlight the importance of glucose as an additional factor in promoting Ps. aeruginosa growth and respiratory infection in CF disease.     

Unraveling the complex genetic model for cystic fibrosis: pleiotropic effects of modifier genes on early cystic fibrosis-related morbidities

The existence of pleiotropy in disorders with multi-organ involvement can suggest therapeutic targets that could ameliorate overall disease severity. Here we assessed pleiotropy of modifier genes in cystic fibrosis (CF). CF, caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, affects the lungs, liver, pancreas and intestines. However, modifier genes contribute to variable disease severity across affected organs, even in individuals with the same CFTR genotype. We sought to determine whether SLC26A9, SLC9A3 and SLC6A14, that contribute to meconium ileus in CF, are pleiotropic for other early-affecting CF co-morbidities. In the Canadian CF population, we assessed evidence for pleiotropic effects on (1) pediatric lung disease severity (n = 815), (2) age at first acquisition of Pseudomonas aeruginosa (P. aeruginosa) (n = 730), and (3) prenatal pancreatic damage measured by immunoreactive trypsinogen (n = 126). A multiple-phenotype analytic strategy assessed evidence for pleiotropy in the presence of phenotypic correlation. We required the same alleles to be associated with detrimental effects. SLC26A9 was pleiotropic for meconium ileus and pancreatic damage (p = 0.002 at rs7512462), SLC9A3 for meconium ileus and lung disease (p = 1.5 × 10^?6 at rs17563161), and SLC6A14 for meconium ileus and both lung disease and age at first P. aeruginosa infection (p = 0.0002 and p = 0.006 at rs3788766, respectively). The meconium ileus risk alleles in SLC26A9, SLC9A3 and SLC6A14 are pleiotropic, increasing risk for other early CF co-morbidities. Furthermore, co-morbidities affecting the same organ tended to associate with the same genes. The existence of pleiotropy within this single disorder suggests that complementary therapeutic strategies to augment solute transport will benefit multiple CF-associated tissues.     

Influence of the cystic fibrosis transmembrane conductance regulator on expression of lipid metabolism-related genes in dendritic cells

Background

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Infections of the respiratory tract are a hallmark in CF. The host immune responses in CF are not adequate to eradicate pathogens, such as P. aeruginosa. Dendritic cells (DC) are crucial in initiation and regulation of immune responses. Changes in DC function could contribute to abnormal immune responses on multiple levels. The role of DC in CF lung disease remains unknown.

Methods

This study investigated the expression of CFTR gene in bone marrow-derived DC. We compared the differentiation and maturation profile of DC from CF and wild type (WT) mice. We analyzed the gene expression levels in DC from naive CF and WT mice or following P. aeruginosa infection.

Results

CFTR is expressed in DC with lower level compared to lung tissue. DC from CF mice showed a delayed in the early phase of differentiation. Gene expression analysis in DC generated from naive CF and WT mice revealed decreased expression of Caveolin-1 (Cav1), a membrane lipid raft protein, in the CF DC compared to WT DC. Consistently, protein and activity levels of the sterol regulatory element binding protein (SREBP), a negative regulator of Cav1 expression, were increased in CF DC. Following exposure to P. aeruginosa, expression of 3β-hydroxysterol-Δ7 reductase (Dhcr7) and stearoyl-CoA desaturase 2 (Scd2), two enzymes involved in the lipid metabolism that are also regulated by SREBP, was less decreased in the CF DC compared to WT DC.

Conclusion

These results suggest that CFTR dysfunction in DC affects factors involved in membrane structure and lipid-metabolism, which may contribute to the abnormal inflammatory and immune response characteristic of CF.     

Genomic, Tissue Expression, and Protein Characterization of pCLCA1, a Putative Modulator of Cystic Fibrosis in the Pig

Recent studies have identified members of the CLCA (chloride channels, calcium-activated) gene family as potential modulators of the cystic fibrosis (CF) phenotype, but differences between the human and murine CLCA genes and proteins may limit the use of murine CF models. Recently established pig models of CF are expected to mimic the human disease more closely than the available mouse models do. Here, we characterized the porcine CLCA gene locus, analyzed the expression pattern and protein processing of pCLCA1, and compared it to its human ortholog, hCLCA1. The porcine CLCA gene family is located on chromosome 4q25, with a broad synteny with the human and murine clca gene loci, except for a pig-specific gene duplication of pCLCA4. Using pCLCA1-specific antibodies, the protein was immunohistochemically localized in mucin-producing cells, including goblet cells and mucinous glands in the respiratory and alimentary tracts. Similar to hCLCA1, biochemical characterization of pCLCA1 identified a secreted soluble protein that could serve as an extracellular signaling molecule or functional constituent of the protective mucous layers. The results suggest that pCLCA1 shares essential characteristics of hCLCA1, supporting the pig model as a promising tool for studying the modulating role of pCLCA1 in the complex pathology of CF. (J Histochem Cytochem 57:1169–1181, 2009)     

Pathophysiologic consequences following inhibition of a CFTR-dependent developmental cascade in the lung

Background  

Examination of late gestation developmental genes in vivo may be limited by early embryonic lethality and compensatory mechanisms. This problem is particularly apparent in evaluating the developmental role of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in the cystic fibrosis (CF) phenotype. A previously described transient in utero knockout (TIUKO) technology was used to address the developmental role of CFTR in the rat lung.     

Cystic-fibrosis-like disease unrelated to the cystic fibrosis transmembrane conductance regulator

Cystic fibrosis (CF) is considered to be a monogenic disease caused by molecular lesions within the cystic fibrosis transmembrane conductance regulator (CFTR) gene and is diagnosed by elevated sweat electrolytes. We have investigated the clinical manifestations of cystic fibrosis, CFTR genetics and electrophysiology in a sibpair in which the brother is being treated as having CF, whereas his sister is asymptomatic. The diagnosis of CF in the index patient is based on highly elevated sweat electrolytes in the presence of CF-related pulmonary symptoms. The investigation of chloride conductance in respiratory and intestinal tissue by nasal potential difference and intestinal current measurements, respectively, provides no evidence for CFTR dysfunction in the siblings who share the same CFTR alleles. No molecular lesion has been identified in the CFTR gene of the brother. Findings in the investigated sibpair point to the existence of a CF-like disease with a positive sweat test without CFTR being affected. Other factors influencing sodium or chloride transport are likely to be the cause of the symptoms in the patient described. Received: 25 August 1997 / Accepted: 20 January 1998     

<Emphasis Type="Italic">Exophiala dermatitidis</Emphasis> Revealing Cystic Fibrosis in Adult Patients with Chronic Pulmonary Disease

Cystic fibrosis (CF) is a genetic inherited disease due to mutations in the gene cystic fibrosis transmembrane conductance regulator (CFTR). Because of the huge diversity of CFTR mutations, the CF phenotypes are highly heterogeneous, varying from typical to mild form of CF, also called atypical CF. These atypical features are more frequently diagnosed at adolescence or adulthood, and among clinical signs and symptoms leading to suspect a mild form of CF, colonization or infection of the respiratory tract due to well-known CF pathogens should be a warning signal. Exophiala dermatitidis is a melanized dimorphic fungus commonly detected in respiratory specimens from CF patients, but only very rarely from respiratory specimens from non-CF patients. We described here two cases of chronic colonization of the airways by E. dermatitidis, with recurrent pneumonia and hemoptysis in one patient, which led clinicians to diagnose mild forms of CF in these elderly patients who were 68- and 87-year-old. These cases of late CF diagnosis suggest that airway colonization or respiratory infections due to E. dermatitidis in patients with bronchiectasis should led to search for a mild form of CF, regardless of the age and associated symptoms. On a broader level, in patients with chronic respiratory disease and recurrent pulmonary infections, an allergic bronchopulmonary mycosis or an airway colonization by CF-related fungi like E. dermatitidis or some Aspergillus, Scedosporium or Rasamsonia species, should be considered as potential markers of atypical CF and should led clinicians to conduct investigations for CF diagnosis.     

Swimming Motility in a Longitudinal Collection of Clinical Isolates of Burkholderia cepacia Complex Bacteria from People with Cystic Fibrosis

Chronic bacterial lung infections in cystic fibrosis (CF) are the leading cause of morbidity and mortality. While a range of bacteria are known to be capable of establishing residence in the CF lung, only a small number have a clearly established link to deteriorating clinical status. The two bacteria with the clearest roles in CF lung disease are Pseudomonas aeruginosa and bacteria belonging to the Burkholderia cepacia complex (BCC). A number of common adaptations by P. aeruginosa strains to chronic lung infection in CF have been well described. Typically, initial isolates of P. aeruginosa are nonmucoid and display a range of putative virulence determinants. Upon establishment of chronic infection, subsequent isolates ultimately show a reduction in putative virulence determinants, including swimming motility, along with an acquisition of the mucoid phenotype and increased levels of antimicrobial resistance. Infections by BCC are marked by an unpredictable, but typically worse, clinical outcome. However, in contrast to P. aeruginosa infections in CF, studies describing adaptive changes in BCC bacterial phenotype during chronic lung infections are far more limited. To further enhance our understanding of chronic lung infections by BCC bacteria in CF, we assessed the swimming motility phenotype in 551 isolates of BCC bacteria from cystic fibrosis (CF) lung infections between 1981 and 2007. These data suggest that swimming motility is not typically lost by BCC during chronic infection, unlike as seen in P. aeruginosa infections. Furthermore, while we observed a statistically significant link between mucoidy and motility, we did not detect any link between motility phenotype and clinical outcome. These studies highlight the need for further work to understand the adaptive changes of BCC bacteria during chronic infection in the CF lung.     

Characterization of Burkholderia cepacia complex from cystic fibrosis patients in China and their chitosan susceptibility

A survey of Burkholderia cepacia complex (Bcc) species was conducted in sputum from cystic fibrosis (CF) patients in China. One hundred and four bacterial isolates were recovered on B. cepacia selective agar and 42 of them were assigned to Bcc by PCR assays. The species composition of the Bcc isolates from CF sputum was analyzed by a combination of recA-restriction fragment length polymorphism assays, species-specific PCR tests and recA gene sequencing. The results revealed that the 42 Bcc isolates belong to B. cepacia, B. cenocepacia and B. contaminans while predominant Bcc species was B. cenocepacia. This is the first report of B. contaminans from CF sputum in China. In addition, results from this study showed that chitosan solution at 10, 25, 50 and 100 μg/ml markedly inhibited the growth of the 16 representative isolates from the three different Bcc species, which indicated that chitosan was a potential bactericide against Bcc bacteria.