Onofrio Laselva1, Paul Dw Eckford2, Claire Bartlett3, Hong Ouyang3, Tarini Na Gunawardena3, Tanja Gonska4, Theo J Moraes5, Christine E Bear6. 1. Programme in Molecular Medicine, Hospital for Sick Children Research Institute, Toronto, Canada; Department of Physiology, University of Toronto, Toronto, Canada. 2. Programme in Molecular Medicine, Hospital for Sick Children Research Institute, Toronto, Canada. 3. Programme in Translational Medicine, Hospital for Sick Children Research Institute, Toronto, Canada. 4. Programme in Translational Medicine, Hospital for Sick Children Research Institute, Toronto, Canada; Department of Paediatrics, University of Toronto, Toronto, Canada. 5. Programme in Translational Medicine, Hospital for Sick Children Research Institute, Toronto, Canada; Department of Paediatrics, University of Toronto, Toronto, Canada. Electronic address: theo.moraes@sickkids.ca. 6. Programme in Molecular Medicine, Hospital for Sick Children Research Institute, Toronto, Canada; Department of Physiology, University of Toronto, Toronto, Canada; Department of Biochemistry, University of Toronto, Toronto, Canada. Electronic address: bear@sickkids.ca.
Abstract
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.
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.
Authors: Onofrio Laselva; Claire Bartlett; Tarini N A Gunawardena; Hong Ouyang; Paul D W Eckford; Theo J Moraes; Christine E Bear; Tanja Gonska Journal: Eur Respir J Date: 2021-06-17 Impact factor: 16.671
Authors: Steven Erwood; Onofrio Laselva; Teija M I Bily; Reid A Brewer; Alexandra H Rutherford; Christine E Bear; Evgueni A Ivakine Journal: Mol Ther Methods Clin Dev Date: 2020-05-12 Impact factor: 6.698
Authors: Onofrio Laselva; Zafar Qureshi; Zhi-Wei Zeng; Evgeniy V Petrotchenko; Mohabir Ramjeesingh; C Michael Hamilton; Ling-Jun Huan; Christoph H Borchers; Régis Pomès; Robert Young; Christine E Bear Journal: iScience Date: 2021-05-15
Authors: Samuel J Bose; Georg Krainer; Demi R S Ng; Mathias Schenkel; Hideki Shishido; Jae Seok Yoon; Peter M Haggie; Michael Schlierf; David N Sheppard; William R Skach Journal: J Cyst Fibros Date: 2020-01-03 Impact factor: 5.482