Figure 6.

Detargeting CFTR expression in basal cells impacts the level of complementation in CF airway epithelia. (A) Diagram of lenti-vector containing CFTR expression cassette in reverse orientation. The PGK promoter (PGKp) drives expression of CFTR with the miR-106b target sequence (4× miR target or mirT) in the 3’UTR. CBA promoter drives expression of Tomato as a reporter gene for viral transduction. pLV-dt/CFTR-Ø is a control vector with no miRT sequence. Box (below) is a legend for each shape in the diagram that highlights the components of these proviral plasmids as described in detail within the Figure 2A legend. (B) Short-circuit current (Isc) measurements of differentiated air-liquid interface cultures seeded with transduced at basal cells. Mock, mock-infected cells. PGK-CFTR-Ø, cells transduced by LV-dt/CFTR-Ø; PGK-CFTR-miRT, cells transduced by LV-dt/CFTR-miRT. Amiloride was used to block ENaC-mediated Na⁺ currents. 4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) was use to inhibit most non-CFTR chloride channel. 3-isobutyl-2-methylxanthine (IBMX) and Forskolin was used for activate CFTR channels. N-(2-naphtalenyl)-(3.5-dibromo-2.4-dihydroxyphenyl)methylene glycine hydrazide (GlyH101) was used to block CFTR. Data show the mean +/-SEM for N = 6 transwells for each condition. (C) Relative quantification of CFTR mRNA normalized to GAPDH mRNA from each sample used in B. For B and C, the statistics used is one-way ANOVA, Tukey’s multiple comparisons test. ****, p < 0.0001. **, p = 0.0025. Data show the mean +/-SEM for N = 3 independent samples for each condition.