(1359-D) Porosome Therapeutics Inc., has Developed Therapy to Rescue Cystic Fibrosis by Reprogramming the Porosome Secretory Machinery

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, leading to lung infections and respiratory failure. A major challenge in treating all CF patients has been the presence of more than 2,000 different CFTR mutations, although the ΔF508 CFTR is the most common, accounting for approximately 70% of all CFTR mutations. CFTR is among the 34 proteins present in the 100 nm porosome secretory machinery, involved in mucin secretion in the human airway epithelia. Reconstitution of functional porosomes having normal CFTR, therefore holds great promise in treating CF with all different types of CFTR mutations. Human airway epithelium 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 a 93% decrease in MUC5AC. Our studies using differentiated Calu-3 3D cultures of human airway epithelial, also demonstrate loss of both chloride and mucus secretion following exposure to both the thiazolidinone CFTR inhibitor 172 and the hydrazide CFTR inhibitor GlyH101. Mass spectrometry and Western Blot analysis of porosomes isolated from WT-CFTR Human Bronchial Epithelial (HBE) Cells and ΔF508-CFTR CF HBE cells, demonstrate a varying loss or gain of several porosome proteins in the ΔF508-CFTR CF HBE cells, including a decrease in the t-SNARE protein SNAP-23 and undetectable levels of the Ras GTPase activating like-protein IQGAP1. These results suggested that mutation in porosome-associated CFTR protein additionally affects other proteins within the porosome secretory machinery, negatively impacting mucus secretion. Hence, to ameliorate defects in mucus secretion in CF, the reconstitution of functional porosomes obtained from WT-CFTR HBE cells into the plasma membrane of ΔF508-CFTR mutant cells was performed. Air liquid interface (ALI) 3D differentiated WT-CFTR and ΔF508-CFTR CF HBE cell cultures were established that mimick normal lung physiology, responding to CFTR inhibitors and CF corrector and modulator drugs Tezacaftor and Ivacaftor. Reconstitution of functional porosome complexes obtained from WT-CFTR HBE cells into the plasma membrane of ΔF508-CFTR CF cells demonstrate that porosome reconstitution restores mucin MUC5B and MUC5AC secretion approximately four-fold more effectively than the currently available CF drugs Tezacaftor and Ivacaftor. On average, porosome reconstitution was able to restore by >40% the secretion of MUC5B in ΔF508-CFTR cells. Similarly, in ΔF508-CFTR CF cells, a 9% and 11% increase in MUC5AC secretion is observed in the presence of Lvacaftor and Tezacaftor respectively, while porosome-reconstitution in the same period demonstrated a significant 43.8% increase (P < 0.05), a near four-fold greater than the two CF drugs. These results show great promise of the porosome-reconstitution therapy in treating cystic fibrosis.