Abstract
The combination therapy of lumacaftor and ivacaftor (Orkambi®) is approved for patients bearing the major cystic fibrosis (CF) mutation: ΔF508. It has been predicted that Orkambi® could treat patients with rarer mutations of similar “theratype”; however, a standardized approach confirming efficacy in these cohorts has not been reported. Here, we demonstrate that patients bearing the rare mutation: c.3700 A>G, causing protein misprocessing and altered channel function—similar to ΔF508‐CFTR, are unlikely to yield a robust Orkambi® response. While in silico and biochemical studies confirmed that this mutation could be corrected and potentiated by lumacaftor and ivacaftor, respectively, this combination led to a minor in vitro response in patient‐derived tissue. A CRISPR/Cas9‐edited bronchial epithelial cell line bearing this mutation enabled studies showing that an “amplifier” compound, effective in increasing the levels of immature CFTR protein, augmented the Orkambi® response. Importantly, this “amplifier” effect was recapitulated in patient‐derived nasal cultures—providing the first evidence for its efficacy in augmenting Orkambi® in tissues harboring a rare CF‐causing mutation. We propose that this multi‐disciplinary approach, including creation of CRISPR/Cas9‐edited cells to profile modulators together with validation using primary tissue, will facilitate therapy development for patients with rare CF mutations.
Highlights
The cystic fibrosis transmembrane conductance regulator (CFTR/ ABCC7) is an ATP- and PKA-dependent chloride channel, regulating chloride and bicarbonate ion flux across apical membranes of polarized epithelial cells in certain tissues
After 30 ns of unrestrained simulation, the loss of secondary structure in bstrands b2 and b3 with varying intensities was evident in multiple simulations of DI1234_R1239-CFTR, while both of these strands remained intact in simulations of WT-CFTR
The root-mean-square deviation (RMSD) of the backbone of each NBD2 residue from the starting conformation of NBD2 as a function of its residue number was compared between WT-CFTR and DI1234_R1239-CFTR systems (Fig 1B)
Summary
The cystic fibrosis transmembrane conductance regulator (CFTR/ ABCC7) is an ATP- and PKA-dependent chloride channel, regulating chloride and bicarbonate ion flux across apical membranes of polarized epithelial cells in certain tissues (e.g., lung, gut, pancreas; Riordan et al, 1989; Bear et al, 1992; Howell et al, 2004; Rowe et al, 2005). The tertiary structure of CFTR is arranged into two membrane-spanning domains (MSDs) with six transmembrane helices (TMs) in each MSD, two intracellular nucleotide binding domains (NBDs), and a regulatory (R) domain (Gadsby et al, 2006). The TMs of MSD1 and MSD2 form the channel pore, while the NBDs form the catalytic heterodimer required for nucleotide-dependent channel gating; the R-domain regulates the gating of this ion channel (Hwang & Kirk, 2013). DF508 is the major mutation, accounting for ~90% of all cases worldwide; there are many additional less frequent CF-causing mutations as documented in the online databases CFTR1 and CFTR2 (see For More Information section; Mickle & Cutting, 2000; Sosnay et al, 2013).
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