Investigating the impact of poly(beta amino) ester-mediated <i>FOXJ1</i> mRNA delivery on differentiation of primary human bronchial epithelial cells

Primary human basal bronchial epithelial cells (HBECs) are an important population of progenitor cells capable of self-renewal and differentiation to maintain airway homeostasis. In air-liquid interface (ALI) culture, HBECs undergo mucociliary differentiation, providing a robust physiologic model to evaluate novel therapeutics such as in vitro-transcribed messenger RNA (IVT-mRNA). However, the impact of IVT-mRNA delivery on the differentiation potential of basal HBECs remains poorly characterised. Poly(beta amino) ester (PBAE) nanoparticles have demonstrated effective airway delivery of IVT-mRNA in various preclinical studies. Here, we aimed to understand the impact of PBAE-mediated mRNA transfection on basal HBEC differentiation at the ALI. We investigated IVT-mRNA encoding the ciliogenesis transcription factor Forkhead box J1 (FOXJ1) as a model tool for transient overexpression in primary basal HBECs and characterised its subsequent impact on epithelial integrity and differentiation at the ALI. PBAE-mediated delivery of FOXJ1 mRNA to submerged primary HBECs resulted in approximately 50% FOXJ1-positive cells and transient upregulation of key ciliogenesis-related genes, including DNALI1 and RSPH9. Following 28 days of differentiation at the ALI, FOXJ1- or reporter mRNA-transfected cultures displayed normal epithelial morphology, with tight junction and differentiation markers, proportions of secretory and ciliated cells, and ciliary ultrastructure comparable to those of non-transfected controls. These data indicate that PBAE-mediated IVT-mRNA delivery can transiently increase encoded protein expression in basal primary HBECs without impeding mucociliary differentiation.