Capsaicin-Loaded Chitosan Nanocapsules for wtCFTR-mRNA Delivery to a Cystic Fibrosis Cell Line.
ABSTRACT: Cystic fibrosis (CF), a lethal hereditary disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene coding for an epithelial chloride channel, is characterized by an imbalanced homeostasis of ion and water transports in secretory epithelia. As the disease is single-gene based, transcript therapy using therapeutic mRNA is a promising concept of treatment in order to correct many aspects of the fatal pathology on a cellular level. Hence, we developed chitosan nanocapsules surface-loaded with wtCFTR-mRNA to restore CFTR function. Furthermore, we loaded the nanocapsules with capsaicin, aiming to enhance the overall efficiency of transcript therapy by reducing sodium hyperabsorption by the epithelial sodium channel (ENaC). Dynamic light scattering with non-invasive back scattering (DLS-NIBS) revealed nanocapsules with an average hydrodynamic diameter of ~200 nm and a Zeta potential of ~+60 mV. The results of DLS-NIBS measurements were confirmed by asymmetric flow field-flow fractionation (AF4) with multidetection, while transmission electron microscopy (TEM) images confirmed the spherical morphology and size range. After stability measurements showed that the nanocapsules were highly stable in cell culture transfection medium, and cytotoxicity was ruled out, transfection experiments were performed with the CF cell line CFBE41o-. Finally, transepithelial measurements with a new state-of-the-art Ussing chamber confirmed successfully restored CFTR function in transfected cells. This study demonstrates that CS nanocapsules as a natural and non-toxic delivery system for mRNA to target cells could effectively replace risky vectors for gene delivery. The nanocapsules are not only suitable as a transcript therapy for treatment of CF, but open aspiring possibilities for safe gene delivery in general.
Project description:Cystic fibrosis (CF), a most deadly genetic disorder, is caused by mutations of CF transmembrane receptor (CFTR) - a chloride channel present at the surface of epithelial cells. In general, two steps have to be involved in treatment of the disease: correction of cellular defects and potentiation to further increase channel opening. Consequently, a combinatorial simultaneous treatment with two drugs with different mechanisms of action, lumacaftor and ivacaftor, has been recently proposed. While lumacaftor is used to correct p.Phe508del mutation (the loss of phenylalanine at position 508) and increase the amount of cell surface-localized CFTR protein, ivacaftor serves as a CFTR potentiator that increases the open probability of CFTR channels. Since the main organ that is affected by cystic fibrosis is the lung, the delivery of drugs directly to the lungs by inhalation has a potential to enhance the efficacy of the treatment of CF and limit adverse side effects upon healthy tissues and organs. Based on our extensive experience in inhalation delivering of drugs by different nanocarriers, we selected nanostructured lipid carriers (NLC) for the delivery both drugs directly to the lungs by inhalation and tested NLC loaded with drugs in vitro (normal and CF human bronchial epithelial cells) and in vivo (homozygote/homozygote bi-transgenic mice with CF). The results show that the designed NLCs demonstrated a high drug loading efficiency and were internalized in the cytoplasm of CF cells. It was found that NLC-loaded drugs were able to restore the expression and function of CFTR protein. As a result, the combination of lumacaftor and ivacaftor delivered by lipid nanoparticles directly into the lungs was highly effective in treating lung manifestations of cystic fibrosis.
Project description:Animal models for cystic fibrosis (CF) have contributed significantly to our understanding of disease pathogenesis. Here we describe development and characterization of the first cystic fibrosis rat, in which the cystic fibrosis transmembrane conductance regulator gene (CFTR) was knocked out using a pair of zinc finger endonucleases (ZFN). The disrupted Cftr gene carries a 16 base pair deletion in exon 3, resulting in loss of CFTR protein expression. Breeding of heterozygous (CFTR+/-) rats resulted in Mendelian distribution of wild-type, heterozygous, and homozygous (CFTR-/-) pups. Nasal potential difference and transepithelial short circuit current measurements established a robust CF bioelectric phenotype, similar in many respects to that seen in CF patients. Young CFTR-/- rats exhibited histological abnormalities in the ileum and increased intracellular mucus in the proximal nasal septa. By six weeks of age, CFTR-/- males lacked the vas deferens bilaterally. Airway surface liquid and periciliary liquid depth were reduced, and submucosal gland size was abnormal in CFTR-/- animals. Use of ZFN based gene disruption successfully generated a CF animal model that recapitulates many aspects of human disease, and may be useful for modeling other CF genotypes, including CFTR processing defects, premature truncation alleles, and channel gating abnormalities.
Project description:Cystic fibrosis (CF) is a common genetic disease caused by mutations in the gene coding for cystic fibrosis transmembrane conductance regulator (CFTR). Although CF affects multiple organ systems, chronic bacterial infections and inflammation in the lung are the leading causes of morbidity and mortality in people with CF. Complementation with a functional CFTR gene repairs this defect, regardless of the disease-causing mutation. In this study, we used a gene delivery system termed piggyBac/adenovirus (Ad), which combines the delivery efficiency of an adenoviral-based vector with the persistent expression of a DNA transposon-based vector. We aerosolized piggyBac/Ad to the airways of pigs and observed widespread pulmonary distribution of vector. We quantified the regional distribution in the airways and observed transduction of large and small airway epithelial cells of non-CF pigs, with ?30-50% of surface epithelial cells positive for GFP. We transduced multiple cell types including ciliated, non-ciliated, basal, and submucosal gland cells. In addition, we phenotypically corrected CF pigs following delivery of piggyBac/Ad expressing CFTR as measured by anion channel activity, airway surface liquid pH, and bacterial killing ability. Combining an integrating DNA transposon with adenoviral vector delivery is an efficient method for achieving functional CFTR correction from a single vector administration.
Project description:Cystic Fibrosis (CF) is an autosomal recessive disease caused by mutations in CF transmembrane conductance regulator (CFTR), resulting in defective anion transport. Regardless of the disease-causing mutation, gene therapy is a strategy to restore anion transport to airway epithelia. Indeed, viral vector-delivered CFTR can complement the anion channel defect. In this proof-of-principle study, functional in vivo CFTR channel activity was restored in the airways of CF pigs using a feline immunodeficiency virus-based (FIV-based) lentiviral vector pseudotyped with the GP64 envelope. Three newborn CF pigs received aerosolized FIV-CFTR to the nose and lung. Two weeks after viral vector delivery, epithelial tissues were analyzed for functional correction. In freshly excised tracheal and bronchus tissues and cultured ethmoid sinus cells, we observed a significant increase in transepithelial cAMP-stimulated current, evidence of functional CFTR. In addition, we observed increases in tracheal airway surface liquid pH and bacterial killing in CFTR vector-treated animals. Together, these data provide the first evidence to our knowledge that lentiviral delivery of CFTR can partially correct the anion channel defect in a large-animal CF model and validate a translational strategy to treat or prevent CF lung disease.
Project description:Cystic Fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and CF patients require life-long treatment. Although CFTR modulators show a great potential for treating most CF patients, some individuals may not tolerate the treatment. In addition, there is no effective therapy for patients with some rare CFTR mutations, such as class I CF mutations, which lead to a lack of CFTR protein production. Therefore, other therapeutic strategies, such as gene therapy, have to be investigated. Currently, immune responses to gene therapy vectors and transgene products are a major obstacle to applying CF gene therapy to clinical applications. In this study, we examined the effects of cyclophosphamide on the modulation of host immune responses and for the improvement of the CFTR transgene expression in the repeated delivery of helper-dependent adenoviral (HD-Ad) vectors to mouse lungs. We have found that cyclophosphamide significantly decreased the expression of T cell genes, such as CD3 (cluster of differentiation 3) and CD4, and reduced their infiltration into mouse lung tissues. We have also found that the levels of the anti-adenoviral antibody and neutralizing activity as well as B-cell infiltration into the mouse lung tissues were significantly reduced with this treatment. Correspondingly, the expression of the human CFTR transgene has been significantly improved with cyclophosphamide administration compared to the group with no treatment. These data suggest that the sustained expression of the human CFTR transgene in mouse lungs through repeated vector delivery can be achieved by transient immunosuppression.
Project description:<h4>Background</h4>Treatments designed to correct cystic fibrosis transmembrane conductance regulator (CFTR) defects must first be evaluated in preclinical experiments in the mouse model of cystic fibrosis (CF). Mice nasal mucosa mimics the bioelectric defect seen in humans. The use of nasal potential difference (V(TE)) to assess ionic transport is a powerful test evaluating the restoration of CFTR function. Nasal V(TE) in CF mice must be well characterized for correct interpretation.<h4>Methods</h4>We performed V(TE) measurements in large-scale studies of two mouse models of CF--B6;129 cftr knockout and FVB F508del-CFTR--and their respective wild-type (WT) littermates. We assessed the repeatability of the test for cftr knockout mice and defined cutoff points distinguishing between WT and F508del-CFTR mice.<h4>Results</h4>We determined the typical V(TE) values for CF and WT mice and demonstrated the existence of residual CFTR activity in F508del-CFTR mice. We characterized intra-animal variability in B6;129 mice and defined the cutoff points for F508del-CFTR chloride secretion rescue. Hyperpolarization of more than -2.15 mV after perfusion with a low-concentration Cl(-) solution was considered to indicate a normal response.<h4>Conclusions</h4>These data will make it possible to interpret changes in nasal V(TE) in mouse models of CF, in future preclinical studies.
Project description:BACKGROUND: In airway epithelial cells, calcium mobilization can be elicited by selective autocrine and/or paracrine activation of apical or basolateral membrane heterotrimeric G protein-coupled receptors linked to phospholipase C (PLC) stimulation, which generates inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG) and induces Ca2+ release from endoplasmic reticulum (ER) stores. METHODS: In the present study, we monitored the cytosolic Ca2+ transients using the UV light photolysis technique to uncage caged Ca2+ or caged IP3 into the cytosol of loaded airway epithelial cells of cystic fibrosis (CF) and non-CF origin. We compared in these cells the types of Ca2+ receptors present in the ER, and measured their Ca2+ dependent activity before and after correction of F508del-CFTR abnormal trafficking either by low temperature or by the pharmacological corrector miglustat (N-butyldeoxynojirimycin). RESULTS: We showed reduction of the inositol 1,4,5-trisphosphate receptors (IP3R) dependent-Ca2+ response following both correcting treatments compared to uncorrected cells in such a way that Ca2+ responses (CF+treatment vs wild-type cells) were normalized. This normalization of the Ca2+ rate does not affect the activity of Ca2+-dependent chloride channel in miglustat-treated CF cells. Using two inhibitors of IP3R1, we observed a decrease of the implication of IP3R1 in the Ca2+ response in CF corrected cells. We observed a similar Ca2+ mobilization between CF-KM4 cells and CFTR-cDNA transfected CF cells (CF-KM4-reverted). When we restored the F508del-CFTR trafficking in CFTR-reverted cells, the specific IP3R activity was also reduced to a similar level as in non CF cells. At the structural level, the ER morphology of CF cells was highly condensed around the nucleus while in non CF cells or corrected CF cells the ER was extended at the totality of cell. CONCLUSION: These results suggest reversal of the IP3R dysfunction in F508del-CFTR epithelial cells by correction of the abnormal trafficking of F508del-CFTR in cystic fibrosis cells. Moreover, using CFTR cDNA-transfected CF cells, we demonstrated that abnormal increase of IP3R Ca2+ release in CF human epithelial cells could be the consequence of F508del-CFTR retention in ER compartment.
Project description:Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel causes the genetic disease cystic fibrosis (CF). Towards the development of transformational drug therapies for CF, we investigated the channel function and action of CFTR potentiators on A561E, a CF mutation found frequently in Portugal. Like the most common CF mutation F508del, A561E causes a temperature-sensitive folding defect that prevents CFTR delivery to the cell membrane and is associated with severe disease.Using baby hamster kidney cells expressing recombinant CFTR, we investigated CFTR expression by cell surface biotinylation, and function and pharmacology with the iodide efflux and patch-clamp techniques.Low temperature incubation delivered a small proportion of A561E-CFTR protein to the cell surface. Like F508del-CFTR, low temperature-rescued A561E-CFTR exhibited a severe gating defect characterized by brief channel openings separated by prolonged channel closures. A561E-CFTR also exhibited thermoinstability, losing function more quickly than F508del-CFTR in cell-free membrane patches and intact cells. Using the iodide efflux assay, CFTR potentiators, including genistein and the clinically approved small-molecule ivacaftor, partially restored function to A561E-CFTR. Interestingly, ivacaftor restored wild-type levels of channel activity (as measured by open probability) to single A561E- and F508del-CFTR Cl(-) channels. However, it accentuated the thermoinstability of both mutants in cell-free membrane patches.Like F508del-CFTR, A561E-CFTR perturbs protein processing, thermostability and channel gating. CFTR potentiators partially restore channel function to low temperature-rescued A561E-CFTR. Transformational drug therapy for A561E-CFTR is likely to require CFTR correctors, CFTR potentiators and special attention to thermostability.
Project description:Cystic Fibrosis (CF), an autosomal recessive genetic disease, is caused by a mutation in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). This mutation reduces the release of chloride ions (Cl-) in epithelial tissues, and hyperactivates the epithelial sodium channels (ENaC) which aid in the absorption of sodium ions (Na+). Consequently, the mucus becomes dehydrated and thickened, making it a suitable medium for microbial growth. CF causes several chronic lung complications like thickened mucus, bacterial infection and inflammation, progressive loss of lung function, and ultimately, death. Until recently, the standard of clinical care in CF treatment had focused on preventing and treating the disease complications. In this review, we have summarized the current knowledge on CF pathogenesis and provided an outlook on the current therapeutic approaches relevant to CF (i.e., CFTR modulators and ENaC inhibitors). The enormous potential in targeting bacterial biofilms using antibiofilm peptides, and the innovative therapeutic strategies in using the CRISPR/Cas approach as a gene-editing tool to repair the CFTR mutation have been reviewed. Finally, we have discussed the wide range of drug delivery systems available, particularly non-viral vectors, and the optimal properties of nanocarriers which are essential for successful drug delivery to the lungs.
Project description:We describe a human nasal epithelial (HNE) organoid model derived directly from patient samples that is well-differentiated and recapitulates the airway epithelium, including the expression of cilia, mucins, tight junctions, the cystic fibrosis transmembrane conductance regulator (CFTR), and ionocytes. This model requires few cells compared to airway epithelial monolayer cultures, with multiple outcome measurements depending on the application. A novel feature of the model is the predictive capacity of lumen formation, a marker of baseline CFTR function that correlates with short-circuit current activation of CFTR in monolayers and discriminates the cystic fibrosis (CF) phenotype from non-CF. Our HNE organoid model is amenable to automated measurements of forskolin-induced swelling (FIS), which distinguishes levels of CFTR activity. While the apical side is not easily accessible, RNA- and DNA-based therapies intended for systemic administration could be evaluated in vitro, or it could be used as an ex vivo biomarker of successful repair of a mutant gene. In conclusion, this highly differentiated airway epithelial model could serve as a surrogate biomarker to assess correction of the mutant gene in CF or other diseases, recapitulating the phenotypic and genotypic diversity of the population.