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Small-molecule eRF3a degraders rescue CFTR nonsense mutations by promoting premature termination codon readthrough
Rhianna E. Lee, … , Adam J. Kimple, Scott H. Randell
Rhianna E. Lee, … , Adam J. Kimple, Scott H. Randell
Published July 28, 2022
Citation Information: J Clin Invest. 2022;132(18):e154571. https://doi.org/10.1172/JCI154571.
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Research Article Pulmonology Article has an altmetric score of 17

Small-molecule eRF3a degraders rescue CFTR nonsense mutations by promoting premature termination codon readthrough

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Abstract

The vast majority of people with cystic fibrosis (CF) are now eligible for CF transmembrane regulator (CFTR) modulator therapy. The remaining individuals with CF harbor premature termination codons (PTCs) or rare CFTR variants with limited treatment options. Although the clinical modulator response can be reliably predicted using primary airway epithelial cells, primary cells carrying rare CFTR variants are scarce. To overcome this obstacle, cell lines can be created by overexpression of mouse Bmi-1 and human TERT (hTERT). Using this approach, we developed 2 non-CF and 6 CF airway epithelial cell lines, 3 of which were homozygous for the W1282X PTC variant. The Bmi-1/hTERT cell lines recapitulated primary cell morphology and ion transport function. The 2 F508del-CFTR cell lines responded robustly to CFTR modulators, which was mirrored in the parent primary cells and in the cell donors’ clinical response. Cereblon E3 ligase modulators targeting eukaryotic release factor 3a (eRF3a) rescued W1282X-CFTR function to approximately 20% of WT levels and, when paired with G418, rescued G542X-CFTR function to approximately 50% of WT levels. Intriguingly, eRF3a degraders also diminished epithelial sodium channel (ENaC) function. These studies demonstrate that Bmi-1/hTERT cell lines faithfully mirrored primary cell responses to CFTR modulators and illustrate a therapeutic approach to rescue CFTR nonsense mutations.

Authors

Rhianna E. Lee, Catherine A. Lewis, Lihua He, Emily C. Bulik-Sullivan, Samuel C. Gallant, Teresa M. Mascenik, Hong Dang, Deborah M. Cholon, Martina Gentzsch, Lisa C. Morton, John T. Minges, Jonathan W. Theile, Neil A. Castle, Michael R. Knowles, Adam J. Kimple, Scott H. Randell

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Figure 3

The eRF3a degrader CC-90009 rescues W1282X-CFTR in a panel of cell lines and parent primary cells.

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The eRF3a degrader CC-90009 rescues W1282X-CFTR in a panel of cell lines...
(A–C) TECC-24 measurements of the UNCX2T cell line (W1282X/W1282X) treated with 0.1% DMSO, 200 μM G418, 0.3 μM SMG1i, 0.1 μM CC-90009, and 3 μM VX-809, alone or in combination as indicated. Acute addition of the potentiator 10 μM VX-770 is indicated by an arrow. (A) TECC-24 tracing representing 3–4 replicates. (B and C) ΔIeq in response to FSK (B) and CFTRinh-172 (C). Data were analyzed using ordinary linear models. n = 3–4. (D–I) TECC-24 measurements of a panel of W1282X/W1282X cell lines and parent cells treated with 0.1% DMSO or 0.1 μM CC-90009. (D and E) TECC-24 tracing of the UNCX2T cell line (D) and parent cells (E). Tracings are representative of the W1282X/W1282X panel containing 3 cell donors with 6 replicates per donor. (F–I) ΔIeq in response to FSK (F) and CFTRinh-172 (G), benazmil (H), and basal Ieq (I). Data were analyzed using a linear mixed-effects model with the donor as a random effect factor. n = 6 per donor. Post hoc comparisons were performed using the general linear hypothesis test. All data are presented as the mean ± SD. ***P < 0.001.

Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)

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