GANGLIOSIDE GM1 AS ADJUVANT FOR ORKAMBI® THERAPY TO RESTORE PLASMA MEMBRANE STABILITY AND FUNCTION OF F508DEL-CFTR

Cystic fibrosis (CF) is the most common, fatal genetic disease in the Caucasian population caused by loss of function mutations in gene encoding for the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is expressed at the apical surface of epithelial cells of different organs, such as: lungs, pancreas, gut, and testes. For this reason even if in CF the pulmonary manifestations are the most severe, CF is considered a multi-system disease, which affects several bodily districts. The new challenge for the CF therapy is based on the development of small molecules able to rescue the function of the mutated CFTR. Many pharmacological agents have been designed to increase the surface level of mutated CFTR (correctors), as well as its plasma membrane (PM) activity (potentiators). Recently, combined therapy that includes a corrector of the CFTR folding (lumacaftor or VX-809) and a potentiator of the channel activity (ivacaftor or VX-770) called Orkambi®, was approved for CF patients homozygous for the deletion of phenylalanine at position 508 (F508del), the most common CF-causing mutation. Unfortunately, clinical studies revealed that the effects of Orkambi® on lung function were modest, due to low stability of rescued F508del-CFTR at the PM level. Indeed, many factors contribute to PM CFTR stability, including its compartmentalization in PM macromolecular complexes composed of phospholipids, sphingolipids, with particular regards for monosialoganglioside 1 (GM1), and scaffolding proteins such as ezrin and NHERF-1. Interestingly, it has been proved that in bronchial epithelial cells the lack of CFTR in the cell PM, such as in the case of the patients carrying the mutation F508del, is associated with a decreased content of GM1. By performing photolabelling experiments, I demonstrated for the first time that GM1 and CFTR at PM level reside in the same microdomain, suggesting a direct interaction between them. Then I investigated on the potential effect of the exogenous administration of ganglioside GM1 on the PM stabilization and function of F508del-CFTR rescued by Orkambi® treatment. In particular, I proved that in CF bronchial epithelial cells GM1 antagonizes the negative effect of VX-770, increasing F508del-CFTR maturation and its channel activity by the recruitment of the scaffolding proteins NHERF-1 and ezrin. Taken together the results obtained during my PhD project pointed out the role of GM1 as possible adjuvant to Orkambi® therapy to restore the function of F508del-CFTR.