Engineering Human Bronchial Epithelial Cells for Cystic Fibrosis Cell Therapy

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ABSTRACT/PROJECT SUMMARY Cystic fibrosis (CF) is a genetic disease in which the cystic fibrosis transmembrane regulator (CFTR) protein is absent or dysfunctional, leading to the buildup of viscous mucous, impaired mucociliary transport, chronic infection and inflammation, and eventually mortality. The cause of CF is challenging to treat, with over 300 known disease-causing protein variants. Cell therapy offers a mutation-blind treatment option; however, cell engraftment into the highly resistive airway epithelium is ineffective and requires extensive airway injury. Approaches to facilitate cellular movement across tight junction barriers and to direct delivered cells to the stem cell niche may be an alternative. Though challenging in the cell therapy model, cellular translocation across epithelial barriers is not without biological precedent. Neutrophils readily migrate into the air space by chemotaxis, the directed migration of cells in response to a chemical stimulant. This motility is initiated when chemokine receptors on the neutrophil’s surface bind target chemokines. In the lungs, chemokines are produced by basal cells, the stem cells of the upper airways and chief residents of the stem cell niche. This process is exceptionally relevant in CF, where bacterial colonization is chronic and the immune response runs rampant. Though chemotaxis is well studied in neutrophils and other immune cells, it is unknown if artificial expression of chemokine receptors in non-immune primary cells would enable them to chemotax through tight junction barriers. Preliminary results from our lab indicate that primary human bronchial epithelial cells (HBECs) engineered to express the chemokine receptor CXCR1 chemotax in the presence of an interleukin-8 (IL8) gradient. As such, our central hypothesis is that engineering HBECs to express chemokine receptors will promote directed migration toward the chemokine source (i.e., the stem cell niche), thus improving the efficiency of cell engraftment. To test this hypothesis, CXCR1 expression will be optimized in HBECs using diverse lentiviral promoters. CXCR1 abundance will be quantified by western blot, and directional migration will be measured in two- and three-dimensional chemotaxis assays. Canonical chemotaxis pathway members will be quantified by western blot and fluorescent reporter assays. The goal of directing exogenously delivered cells toward the stem cell niche is ultimately to increase long-term cell engraftment with minimal injury requirements. Thus, the engraftment efficiency of engineered HBECs will be evaluated in vitro by quantifying chimerism after delivery to air-liquid interface cultures stimulated to produce IL8 by an inflammatory challenge. Finally, engraftment efficiency will be assessed in vivo by delivering engineered airway cells to the murine trachea and by tracking real-time cell migration by two-photon microscopy. Engineering cells to home to the stem cell niche with minimal injury requirements would be a monumental step toward increasing the safety and efficacy of CF cell therapy. The proof-of-concept studies proposed here will bring us one step closer to this goal.