Figure 2.

EF1α drives higher transgene expression in primary human airway epithelial cells compared to PGK

(A and B) Basal progenitor cells from five human donors without CF (non-CF) donors were transduced with lentiviral vectors HIV-PGK-GFP or HIV-EF1α-GFP at MOI 0.04, 0.4, or 4. GFP⁺ cells and mean fluorescence intensity (MFI) were quantified by flow cytometry (A), 3−5 days post-transduction and (B) after 4 weeks of differentiation. No significant difference was observed in the number of GFP⁺ cells transduced by either vector at any dose. A significant increase in MFI was observed in cells transduced with HIV-EF1α-GFP compared to HIV-PGK-GFP at MOI 4 at both time points (∗p < 0.0006, ∗∗p < 0.002). Similarly, basal cells from four human CF donors were transduced with lentiviral vectors HIV-PGK-WTCFTR, HIV-EF1α-WTCFTR, or HIV-PGK-GFP. (C) After 4 weeks of differentiation under air-liquid interface conditions, the number of remaining transduced cells was estimated through quantification of GFP⁺ cells by flow cytometry. (D) Transepithelial Cl⁻ current was measured in Ussing chambers. ENaC and non-CFTR chloride channels were inhibited with sequential addition of amiloride and 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS), respectively, prior to activation of CFTR channels with cyclic AMP (cAMP) agonists forskolin and 3-isobutyl-1-methylxanthine (F&I). CFTR-specific current was verified by addition of CFTR inhibitor GlyH-101 (GlyH). (E) The short circuit current change (ΔI_SC) in response to F&I and GlyH was calculated. No significant difference between the two vectors was observed at any dose. Current measurements in cultures from one donor in two treatment groups (HIV-EF1α-WTCFTR MOI 0.04 and 4) could not be completed due to unsuccessful differentiation. Points on the graph represent the average of 1−3 epithelia. Each donor is represented by a unique symbol. Mean ± SE is shown.