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

Abstract

<h3>Introduction</h3> 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. <h3>Methods</h3> 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). <h3>Results</h3> Transduction efficiency was generally &lt;1% of cells (%GFP area: 0.46±0.05%, Flow cytometry: 0.60±0.14%, n=9/group). There was no difference in transduction efficiency between HC and CF ALIs. Sendai virus, which transduces lung epithelium with high efficiency <i>in vivo</i> also only produced low level transduction in these cultures (HC: 2.2±1.1%, CF: 0.72±0.13%, n=6/group). 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&lt;0.05) in transduced CF ALIs compared to CF controls (CF-CFTR: 3.8±0.7 µA/cm², CF: 1.16±0.3 µA/cm², n=14–19/group). Corrected CF ALI cultures achieved ~40% of the HC response (CF-CFTR: 3.8±0.73 µA/cm², HC: 8.9±1.4 µA/cm²). <h3>Conclusion</h3> These data suggest that <i>ex vivo</i> transduction efficiency of differentiated human ALIs is low and may not reflect the <i>in vivo</i> 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.