Functional rescue of c.3846G>A (W1282X) in patient-derived nasal cultures achieved by inhibition of nonsense mediated decay and protein modulators with complementary mechanisms of action

Background: The nonsense mutation, c.3846G>A (aka: W1282X-CFTR) leads to a truncated transcript that is susceptible to nonsense-mediated decay (NMD) and produces a shorter protein that is unstable and lacks normal channel activity in patient-derived tissues. However, if overexpressed in a heterologous expression system, the truncated mutant protein has been shown to mediate CFTR channel function following the addition of potentiators. In this study, we asked if a quadruple combination of small molecules that together inhibit nonsense mediated decay, stabilize both halves of the mutant protein and potentiate CFTR channel activity could rescue the functional expression of W1282X-CFTR in patient derived nasal cultures. Methods: We identified the CFTR domains stabilized by corrector compounds supplied from AbbVie using a fragment based, biochemical approach. Rescue of the channel function of W1282X.-CFTR protein by NMD inhibition and small molecule protein modulators was studied using a bronchial cell line engineered to express W1282X and in primary nasal epithelial cultures derived from four patients homozygous for this mutation. Results: We confirmed previous studies showing that inhibition of NMD using the inhibitor: SMG1i, led to an increased abundance of the shorter transcript in a bronchial cell line. Interestingly, on top of SMG1i, treatment with a combination of two new correctors developed by Galapagos/AbbVie (AC1 and AC2-2, separately targeting either the first or second half of CFTR and promoting assembly, significantly increased the potentiated channel activity by the mutant in the bronchial epithelial cell line and in patient-derived nasal epithelial cultures. The average rescue effect in primary cultures was approximately 50% of the regulated chloride conductance measured in non-CF cultures. Conclusions: These studies provide the first in-vitro evidence in patient derived airway cultures that the functional defects incurred by W1282X, has the potential to be effectively repaired pharmacologically. (C) 2019 The Authors. Published by Elsevier B.V. on behalf of European Cystic Fibrosis Society.