S67 Low levels of lentivirus-mediated CFTR gene transfer are sufficient to generate ion transport correction in air-liquid interface cultures from cystic fibrosis patients

Introduction We have developed a lentiviral vector pseudotyped with the F and HN proteins from Sendai virus (rSIV.F/HN) for cystic fibrosis (CF) gene therapy and are now progressing towards a first-in-man clinical trial. Here we assessed the transduction efficiency of rSIV.F/HN expressing EGFP in human bronchial epithelial cells from healthy control (HC) and CF donors grown in air-liquid interface culture (ALI). We also assessed the degree of correction of ion transport by rSIV.F/HN-CFTR in this model. Methods Fully differentiated ALIs (MucilAir, Epithelix) were transduced with rSIV.F/HN-EGFP or Sendai virus (SeV)-GFP and GFP expression was quantified at multiple time points using fluorescence microscopy or flow cytometry. The ion transport in HC, CF, and CF ALIs transduced with rSIV.F/HN-CFTR was measured at 7 days in Ussing chambers (stepwise protocol: chloride buffer as baseline, 100 μM amiloride, 100 μM DIDS, low chloride, 10 μM forskolin/100 μM IBMX, 30 μM ΔCFTR inhibitor-172). Results Transduction efficiency was generally In Ussing chambers, there was no difference in baseline short circuit current between HC and CF ALIs, while forskolin/IBMX-mediated chloride secretion was significantly higher in HC samples compared to CF (HC: 8.9±1.4 µA/cm2, CF: 1.16±0.33 µA/cm2, n=14–17/group). We then assessed whether transduction of CF ALIs with rSIV.F/HN-CFTR was able to correct the chloride transport defect. Chloride transport increased significantly (p Conclusion These data suggest that ex vivo transduction efficiency of differentiated human ALIs is low and may not reflect the in vivo performance of gene transfer agents. However, even at this low transduction efficiency, functional correction of ~40% of chloride transport was achieved in CF patient-derived ALI cultures following transduction with rSIV.F/HN-CFTR.