A fundamental challenge for cystic fibrosis (CF) gene therapy is ensuring sufficient transduction of airway epithelial cells to achieve therapeutic correction. Vehicles such as lysophosphatidylcholine (LPC), EGTA, perfluorocarbon, or methylcellulose have been used to enhance gene transfer of Ad, AAV or lentiviral vectors. However, advancing these formulations to a clinical setting adds an additional layer to translating a gene therapy therapeutic. To this note, we screened FDA approved reagents for their ability to enhance viral vector transduction of primary human airway epithelial cells and identified hypertonic saline as a candidate that conferred a clear benefit. Hypertonic saline (3-7%) is frequently administered as an agent to enhance mucociliary clearance in people with CF. Hypertonic saline transiently disrupts epithelial cell tight junctions, but its ability to improve gene transfer has not been investigated. Here we asked if increasing NaCl tonicity in the vector formulation enhances the transduction efficiency of 3 gene therapy vectors: adenovirus, AAV, and lentiviral vectors. We observed that vectors formulated in vitro with 3-5% NaCl exhibited markedly increased transduction for all 3 platforms, leading to phenotypic correction of the anion channel defect in primary cultures of human CF epithelial cells. The NaCl-enhanced transduction was enhanced by ionic strength but not osmolarity, or pH. Focused Ad entry studies suggest viral entry remains receptor dependent and requires low pH endosomal escape. Gene transfer in vivo was also enhanced in mouse and pig airways by formulating vectors in NaCl (5% or 7%). Increasing saline tonicity has the potential to increase the viral delivery efficacy with a reduced amount of vector. These studies open the door to many possibilities for other genetic lung diseases, far beyond CF.

Learning Objectives:

• Identify the effects of hypertonic saline on transduction efficiency for adenovirus, AAV, and lentiviral vectors in airway epithelial cells.
• Differentiate the impact of ionic strength versus osmolarity and pH on NaCl-enhanced gene transfer.
• Evaluate the potential of hypertonic saline formulations to improve gene therapy outcomes in cystic fibrosis and other genetic lung diseases.