Porosome reconstitution therapy: A biologic rescue from cystic fibrosis

ABSTRACT Cystic fibrosis (CF) is a genetic disorder resulting from mutations in the CF Transmembrane Conductance Regulator (CFTR) gene that codes for a chloride transporting channel at the cell plasma membrane. In CF, highly viscous mucus is secreted in the airways preventing its clearance, leading to lung infections and respiratory failure. A major challenge in treating CF patients has been the presence of more than 2,000 different CFTR mutations or due to the absence of CFTR expression. CFTR is among the 34 major proteins composing the 100 nm porosome secretory machinery in the human airway epithelia, involved in mucin secretion. The airways is coated with a thin film of mucus, composed primarily of mucin MUC5AC and MUC5B. Sputum from patients with CF show a >70% decrease in MUC5B and MUC5AC secretion. Our studies using differentiated 3D cultures of human airway epithelial cell line, also demonstrate loss of both chloride and mucus secretion following exposure to CFTR inhibitors thiazolidinone 172 or the hydrazide GlyH101. Our studies show that human bronchial epithelial (HBE) cells with ΔF508 CFTR mutation, affects nearly a dozen porosome proteins including CFTR. Therefore, we hypothesized that the introduction of normal functional porosomes carrying wild type CFTR into the cell plasma membrane of CF cells would rescue from all forms of CF. Air liquid interface (ALI) 3D differentiated HBE WT-CFTR cells and ΔF508-CFTR CF HBE cell cultures mimicking normal lung physiology, responding to CFTR inhibitors and CF corrector and modulator drugs Tezacaftor, Ivacaftor and TRIKAFTA, was used in the study. Introduction of functional porosome complexes obtained from WT-CFTR HBE cells into the plasma membrane (PM) of ΔF508-CFTR CF cells, was demonstrated by an increase in PM-associated CFTR using Magnify expansion microscopy. Mucin secretion assays demonstrate porosome reconstitution to restore mucin secretion more than twice as effectively as TRIKAFTA. These results are further supported by preliminary nasal potential different studies in ΔF508 mice, where treatment of the nasal passage with porosome isolates from WT-CFTR HBE cells, restore chloride secretion in the nasal passage of mice, a further validation of the highly effective porosome reconstitution therapy for CF.