Role of clathrin- and caveolae-mediated endocytosis in gene transfer mediated by lipo- and polyplexes

Abstract We investigated the effects of inhibitors of clathrin-mediated endocytosis (chlorpromazine and K + depletion) and of caveolae-mediated uptake (filipin and genistein) on internalization of FITC–poly-l-lysine-labeled DOTAP/DNA lipoplexes and PEI/DNA polyplexes by A549 pneumocytes and HeLa cells and on the transfection efficiencies of these complexes with the luciferase gene. Uptake of the complexes was assayed by fluorescence-activated cell sorting. Lipoplex internalization was inhibited by chlorpromazine and K + depletion but unaffected by filipin and genistein. In contrast, polyplex internalization was inhibited by all four inhibitors. We conclude that lipoplex uptake proceeds only by clathrin-mediated endocytosis, while polyplexes are taken up by two mechanisms, one involving caveolae and the other clathrin-coated pits. Transfection by lipoplexes was entirely abolished by blocking clathrin-mediated endocytosis, whereas inhibition of the caveolae pathway had no effect. By contrast, transfection mediated by polyplexes was completely blocked by genistein and filipin but was unaffected by inhibitors of clathrin-mediated endocytosis. Fluorescence colocalization studies with a lysosomal marker, AlexaFluor–dextran, revealed that polyplexes taken up by clathrin-mediated endocytosis are targeted to the lysosomal compartment for degradation, while the polyplexes internalized via caveolae escape this compartment, permitting efficient transfection

Role of Neutrophils in Cystic Fibrosis Lung Disease

Cystic fibrosis (CF) is a genetic syndrome caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene. In CF patients, chief morbidity and mortality are due to pulmonary manifestations. CFTR lack/dysfunction brings an altered ion flux through the airway epithelium and ablation of mucociliary clearance, which in turn ensues in colonization and infection by opportunistic bacterial pathogens and subsequent neutrophil‐dominated inflammation. This response eventually leads to the damage of the lung tissue. A host of inflammatory mediators attract, activate, and reprogramme neutrophils to survive (avoiding apoptosis) and produce a wealth of proteases and radical oxygen species. The protease/antiprotease imbalance and oxidative stress have multiple downstream effects, including impaired mucus clearance, increased and self‐perpetuating inflammation, and impaired immune responses, thus facilitating and fostering bacterial infections. On the other hand, CFTR lack or dysfunction is likely responsible for alterations in neutrophils concerning chemotaxis, phagocytosis, oxidative burst, degranulation, and neutrophil extracellular trap (NET) formation. A good opportunity to reveal new and non‐invasive biomarkers of CF lung disease is the evaluation of circulating neutrophils. Indeed, neutrophil responses are now investigated as outcomes of the aetiological therapies in CF, such as hypertonic saline, antiproteases, CFTR correctors and potentiators

Impact of Lentiviral Vector-Mediated Transduction on the Tightness of a Polarized Model of Airway Epithelium and Effect of Cationic Polymer Polyethylenimine

Lentiviral (LV) vectors are promising agents for efficient and long-lasting gene transfer into the lung and for gene therapy of genetically determined pulmonary diseases, such as cystic fibrosis, however, they have not been evaluated for cytotoxicity and impact on the tightness of the airway epithelium. In this study, we evaluated the transduction efficiency of a last-generation LV vector bearing Green Fluorescent Protein (GFP) gene as well as cytotoxicity and tight junction (TJ) integrity in a polarized model of airway epithelial cells. High multiplicities of infection (MOI) showed to be cytotoxic, as assessed by increase in propidium iodide staining and decrease in cell viability, and harmful for the epithelial tightness, as demonstrated by the decrease of transepithelial resistance (TER) and delocalization of occludin from the TJs. To increase LV efficiency at low LV:cell ratio, we employed noncovalent association with the polycation branched 25 kDa polyethylenimine (PEI). Transduction of cells with PEI/LV particles resulted in 2.5–3.6-fold increase of percentage of GFP-positive cells only at the highest PEI:LV ratios (1×107 PEI molecules/transducing units with 50 MOI LV) as compared to plain LV. At this dose PEI/LV transduction resulted in 6.5 ± 2.4% of propidium iodide-positive cells. On the other hand, PEI/LV particles did not determine any alteration of TER and occludin localization. We conclude that PEI may be useful for improving the efficiency of gene transfer mediated by LV vectors in airway epithelial cells, in the absence of high acute cytotoxicity and alteration in epithelial tightness

A VSV-G Pseudotyped Last Generation Lentiviral Vector Mediates High Level and Persistent Gene Transfer in Models of Airway Epithelium In Vitro and In Vivo

The aim of this work was to evaluate the efficiency and duration of gene expression mediated by a VSV-G pseudotyped last generation lentiviral (LV) vector. We studied LV efficiency in ex-vivo models of respiratory epithelial cells, obtained from bronchial biopsies and nasal polyps, by GFP epifluorescence and cytofluorimetry. In vivo efficiency and persistence of gene expression was investigated by GFP immunohistochemistry and luciferase activity in lung cryosections and homogenates, respectively, upon intranasal and intratracheal administration protocols in C57Bl/6 mice. Both primary bronchial and nasal epithelial cells were transduced up to 70–80% 72 hr after the LV infection. In vivo nasal luciferase expression was increased by lysophosphatidylcholine pre-treatment of the nose. Conversely, the bronchial epithelium was transduced in the absence of any pre-conditioning treatment and luciferase expression lasted for at least 6 months without any decline. We conclude that a last generation LV vector is a promising gene transfer agent in the target organ of genetic and acquired lung diseases, as in the case of cystic fibrosis

Insulin-Like Growth Factor Binding Protein (IGFBP-6) as a Novel Regulator of Inflammatory Response in Cystic Fibrosis Airway Cells

Cystic Fibrosis (CF) patients are prone to contracting bacterial lung infections with opportunistic pathogens, especially Pseudomonas aeruginosa. Prolonged P. aeruginosa infections have been linked to chronic inflammation in the CF lung, whose hallmarks are increased levels of cytokines (i.e., TNF-alpha, IL-1 beta, IL-6) and neutrophil attraction by chemokines, like IL-8. Recently, insulin-like growth factor binding protein 6 (IGFBP-6) has been shown to play a putative role in the immune system and was found at higher levels in the sera and synovial tissue of rheumatoid arthritis patients. Moreover, it has been demonstrated that IGFBP-6 has chemoattractant properties towards cells of the innate (neutrophils, monocytes) and adaptive (T cells) immunity. However, it is not known whether IGFBP-6 expression is dysregulated in airway epithelial cells under infection/inflammatory conditions. Therefore, we first measured the basal IGFBP-6 mRNA and protein levels in bronchial epithelial cells lines (Wt and F508del-CFTR CFBE), finding they both are upregulated in F508del-CFTR CFBE cells. Interestingly, LPS and IL-1 beta+TNF alpha treatments increased the IGFBP-6 mRNA level, that was reduced after treatment with an anti-inflammatory (Dimethyl Fumarate) in CFBE cell line and in patient-derived nasal epithelial cultures. Lastly, we demonstrated that IGFBP-6 reduced the level of pro-inflammatory cytokines in both CFBE and primary nasal epithelial cells, without affecting rescued CFTR expression and function. The addition of a neutralizing antibody to IGFBP-6 increased pro-inflammatory cytokines expression under challenge with LPS. Together, these data suggest that IGFBP-6 may play a direct role in the CF-associated inflammation

Assembly and functional analysis of an S/MAR based episome with the cystic fibrosis transmembrane conductance regulator gene

Improving the efficacy of gene therapy vectors is still an important goal toward the development of safe and efficient gene therapy treatments. S/MAR (scaffold/matrix attached region)-based vectors are maintained extra-chromosomally in numerous cell types, which is similar to viral-based vectors. Additionally, when established as an episome, they show a very high mitotic stability. In the present study we tested the idea that addition of an S/MAR element to a CFTR (cystic fibrosis transmembrane conductance regulator) expression vector, may allow the establishment of a CFTR episome in bronchial epithelial cells. Starting from the observation that the S/MAR vector pEPI-EGFP (enhanced green fluorescence protein) is maintained as an episome in human bronchial epithelial cells, we assembled the CFTR vector pBQ-S/MAR. This vector, transfected in bronchial epithelial cells with mutated CFTR, supported long term wt CFTR expression and activity, which in turn positively impacted on the assembly of tight junctions in polarized epithelial cells. Additionally, the recovery of intact pBQ-S/MAR, but not the parental vector lacking the S/MAR element, from transfected cells after extensive proliferation, strongly suggested that pBQ-S/MAR was established as an episome. These results add a new element, the S/MAR, that can be considered to improve the persistence and safety of gene therapy vectors for cystic fibrosis pulmonary disease