Human NGFR lentiviral particles

Gene transfer to airway epithelial cells is hampered by extracellular (mainly mucus) and cellular (tight junction) barriers. Magnetoresis has been used to increase the residence time of lentiviral vectors (LV) on the cell surface. In this study, magnetoresis was investigated in an airway epithelial cell model that mimics extracellular and cellular barriers. LV-mediated transduction was assessed after polarization of bronchial epithelial cells (H441 line) to filters and dexamethasone (dex) treatment (induction of hemicystosis) with or without magnetoresis. Magnetoresis of unpolarized H441 cells increased transduction at 50 MOI (multiplicity of infection, i.e., transducing units/cell) to 500 MOI in the absence of magnetoresis. Magnetoresis enhanced LV-mediated transduction in mucus layer cells by 20.3-fold. In dexamethasone-induced hemicystosis, LV-mediated transduction efficiency decreased over time. In dome-forming cells, dexamethasone treatment increased zonula occludens-1 (ZO-1) localization at cell borders. Under these experimental conditions, magnetofection significantly increased LV transduction by 5.3-fold. Together, these results suggest that magnetofection can enhance LV-mediated gene transfer to airway epithelial cells in the presence of extracellular (sputum) and cellular (tight junction) barriers, representing a CF-like situation.

H441 cells grown in the presence of 50 nM dexamethasone develop fluid-filled hemicysts, called "domes," that begin to appear from the third day of treatment (Figure 1a). The transduction efficiency of LVs was determined in H441 cultures induced with dexamethasone that were developing domes. In preliminary experiments, the researchers used an MOI of 50 for untreated cells and no ∆NFGR expression was detected. Therefore, transductions were performed at a higher virion-to-cell ratio (i.e., 2000 MOI). With increasing duration of dexamethasone treatment, H441 gradually became more resistant to infection, even at higher MOI of 2000 by using LV–∆NGFR (a lentivirus encoding for nerve growth factor receptor) (Figure 1b). To confirm whether a higher degree of tight junction organization was a feature of dexamethasone-treated H441, the researchers studied transepithelial resistance (TER) and ZO-1 localization in polarized H441 cultures. Figure 1c shows the increase in TER with dexamethasone incubation time, up to 6 days, while the increase in TER without dexamethasone treatment was significantly less pronounced. In H441 cells cultured in the absence of dexamethasone, ZO-1 showed discontinuous junctional staining, not present at every intercellular junction (Figure 1d), whereas its staining was stronger in dexamethasone-treated cells, present at all intercellular boundaries (Figure 1d).

Figure 1. Effect of dexamethasone treatment on dome formation, transepithelial resistance (TER), zonula occludens-1 (ZO-1) organization, and efficiency of LV transduction. (Castellani S, et al., 2016)