Millen, L. | bioRxiv Science

bioRxiv2023

In Vitro Modulator Responsiveness of 655 CFTR Variants Found in People With CF

BackgroundIn 2017, the US Food and Drug Administration initiated expansion of drug labels for the treatment of cystic fibrosis (CF) to include CF transmembrane conductance regulator (CFTR) gene variants based on in vitro functional studies. This study aims to identify CFTR variants that result in increased chloride (Cl-) transport function by the CFTR protein after treatment with the CFTR-modulator combination elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA). These data may benefit people with CF (pwCF) who are not currently eligible for modulator therapies. MethodsPlasmid DNA encoding 655 CFTR variants and wild-type (WT) CFTR were transfected into Fisher Rat Thyroid cells that do not natively express CFTR. After 24 hours of incubation with control or TEZ and ELX, and acute addition of IVA, CFTR function was assessed using the transepithelial current clamp conductance assay. Each variants baseline activity, responsiveness to IVA alone, and responsiveness to the TEZ/ELX/IVA combination were measured in three different laboratories. Western blots were conducted to evaluate CFTR protein maturation and complement the functional data. Results and Conclusions253 variants not currently approved for CFTR modulator therapy showed low baseline activity (<10% of normal CFTR Cl- transport activity). For 152 of these variants, treatment with ELX/TEZ/IVA improved the Cl- transport activity by [≥]10% of normal CFTR function, which is suggestive of clinical benefit. ELX/TEZ/IVA increased CFTR function by [≥]10 percentage points for an additional 140 unapproved variants with [≥]10% but <50% of normal CFTR function at baseline. These findings significantly expand the number of rare CFTR variants for which ELX/TEZ/IVA treatment should result in clinical benefit.

pharmacology and toxicology↗

bioRxiv2021

Role of transmembrane spanning domain 1 in cystic fibrosis transmembrane conductance regulator folding

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein that disrupt its folding pathway. The most common mutation causing CF is a deletion of phenylalanine at position 508 ({Delta}F508). CFTR contains five domains that each form cotranslational structures that interact with other domains as they are produced and folded. CFTR is comprised of two transmembrane spanning domains (TMDs), two nucleotide binding domains (NBDs) and a unique regulatory region (R). The first domain translated, TMD1, forms interdomain interactions with the other domains in CFTR. In TMD1, long intracellular loops extend into the cytoplasm and interact with both NBDs via coupling helices and with TMD2 via transmembrane spans (TMs). We examined mutations in TMD1 to determine the impact on individual domain and multidomain constructs. We found that mutations in a TM span or in the cytosolic ICLs interfere with specific steps in the hierarchical folding of CFTR. TM1 CF-causing mutants, G85E and G91R, directly affect TMD1, whereas most ICL1 and ICL2 mutant effects become apparent in the presence of TMD2. A single mutant in ICL2 worsened CFTR trafficking in the presence of NBD2, supporting its role in the ICL2-NBD2 interface. Mutation of hydrophobic residues in ICL coupling helices tended to increased levels of pre-TMD2 biogenic intermediates but caused ER accumulation in the presence of TMD2. This suggests a tradeoff between transient stability during translation and final structure. NBD2 increased the efficiency of mutant trafficking from the ER, consistent with stabilization of the full-length constructs. While the G85E and G91R mutants in TM1 have immediately detectable effects, most of the studied mutant effects and the {Delta}F508 mutant are apparent after production of TMD2, supporting this intermediate as a major point of recognition by protein quality control.

molecular biology↗