PHARMACOLOGICAL RESCUE OF NONSENSE MUTATIONS IN CYSTIC FIBROSIS

Cystic fibrosis (CF) is caused by loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel that particularly affects the respiratory system, with bacterial infection and severe inflammation. Patients with the most frequent mutation (F508del) or with many types of missense mutations can be efficiently treated with CFTR modulators, namely correctors and potentiators, i.e. pharmacological agents that improve CFTR protein processing and channel activity. However, such treatments are not appropriate by themselves for people carrying nonsense mutations that lead to CFTR protein truncation. We evaluated the efficacy of combinations of drugs targeting at various levels the effects of nonsense mutations: SMG1i to protect CFTR mRNA from nonsense-mediated decay (NMD); G418 and ELX-02, and the eRF3a degrader CC-90009 for protein read-through (RT); correctors VX-809 and VX-445 to promote protein maturation and function, PTI-428 to enhance CFTR protein synthesis. We found that the extent of rescue and sensitivity to the various agents is largely dependent on the type of mutation, with the W1282X as the most responsive mutation to rescue maneuvers. W1282X-CFTR protein expression and function can be largely rescued by NMD suppression with SMG1i and by protein stabilization with the combination of correctors VX-809/VX-445. In contrast, G542X-CFTR required treatment with RT agents and VX-809 but was insensitive to SMG1i. To identify other types of NMD inhibitors, possibly with better characteristics than SMG1i, we carried out a screening of a chemical library of more than 9000 compounds using a functional assay. We identified three novel kinase inhibitors, CC-115, samotolisib, and elimusertib, that act as potent NMD inhibitors. We also evaluated the efficacy of CC90009 on G542X-CFTR in combination with RT agents (ELX-02) and CFTR correctors (VX-809). Treatment resulted in a significant but relatively modest (three-fold) increase in CFTR function. Importantly, the efficacy of the triple drug combination was largely amplified under inflammatory conditions, achieved by exposing the epithelia to IL-4 or to IL-17A/TNF-α. The large rescue of CFTR function was paralleled by the appearance of full-length CFTR protein and by the increase in CFTR mRNA. The effect of inflammatory stimuli on G542X-CFTR could be mediated by enhanced translational RT and/or reduced NMD inhibition. Our results indicate that treatment of CF patients with nonsense mutations requires a precision medicine approach with the design of specific drug combinations for each mutation. Our findings may also lead to the identification of novel targets to correct the effect of nonsense mutations in CF and other genetic diseases. Moreover, and most importantly, our results suggest that pharmacological rescue of CFTR with nonsense mutations could be more effective than expected in vivo due to inflammatory conditions.