Abstract
Gene transfer to airway epithelial cells is hampered by extracellular (mainly mucus) and cellular (tight junctions) barriers. Magnetofection has been used to increase retention time of lentiviral vectors (LV) on the cellular surface. In this study, magnetofection was investigated in airway epithelial cell models mimicking extracellular and cellular barriers. Bronchiolar epithelial cells (H441 line) were evaluated for LV-mediated transduction after polarization onto filters and dexamethasone (dex) treatment, which induced hemicyst formation, with or without magnetofection. Sputum from cystic fibrosis (CF) patients was overlaid onto cells, and LV-mediated transduction was evaluated in the absence or presence of magnetofection. Magnetofection of unpolarized H441 cells increased the transduction with 50 MOI (multiplicity of infection, i.e., transducing units/cell) up to the transduction obtained with 500 MOI in the absence of magnetofection. Magnetofection well-enhanced LV-mediated transduction in mucus-layered cells by 20.3-fold. LV-mediated transduction efficiency decreased in dex-induced hemicysts in a time-dependent fashion. In dome-forming cells, zonula occludens-1 (ZO-1) localization at the cell borders was increased by dex treatment. Under these experimental conditions, magnetofection significantly increased LV transduction by 5.3-fold. In conclusion, these results show that magnetofection can enhance LV-mediated gene transfer into airway epithelial cells in the presence of extracellular (sputum) and cellular (tight junctions) barriers, representing CF-like conditions.
Highlights
The airway epithelium represents the target of gene delivery vectors in inherited (e.g., cystic fibrosis(CF)) and acquired lung diseases [1], extracellular and cellular barriers posit serious limits to the efficiency of this therapeutic approach
Sputum from cystic fibrosis (CF) patients was overlaid onto cells, and lentiviral vectors (LV)-mediated transduction was evaluated in the absence or presence of magnetofection
Investigations made more than 10 years ago [3,4] demonstrated that pathological mucus impedes viral and nonviral gene transfer into airway epithelial cells, only recently has it been shown that the diffusion of both adenovirus and adeno-associated virus is strongly hindered in CF purulent mucus [5]
Summary
The airway epithelium represents the target of gene delivery vectors in inherited (e.g., cystic fibrosis(CF)) and acquired (e.g., asthma) lung diseases [1], extracellular and cellular barriers posit serious limits to the efficiency of this therapeutic approach. The airway mucus is the most important one [2]. Poor gene transfer to the airway epithelium has been attributed to limited cellular uptake across the apical membrane of the lung airway epithelium, inefficient trafficking to the nucleus, vector toxicity, and immunological barriers [6]. Tight junctions (TJs) can represent a formidable barrier to the entry of viruses into the airway epithelial cells. TJs restricts apical entry of viral gene transfer agents into the airway epithelium, since the expression of receptors for viral vectors is more abundant
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