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

Research ArticlePulmonologyTherapeutics Open Access | 10.1172/JCI154571

Rhianna E. Lee,1,2 Catherine A. Lewis,1,3 Lihua He,1 Emily C. Bulik-Sullivan,1,2 Samuel C. Gallant,1 Teresa M. Mascenik,1 Hong Dang,1 Deborah M. Cholon,1 Martina Gentzsch,1,4 Lisa C. Morton,1 John T. Minges,1 Jonathan W. Theile,5 Neil A. Castle,5 Michael R. Knowles,1 Adam J. Kimple,1,6 and Scott H. Randell1,2

1Marsico Lung Institute and Cystic Fibrosis Research Center,

2Department of Cell Biology and Physiology,

3Department of Microbiology and Immunology, and

4Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

5Icagen LLC, Durham, North Carolina, USA.

6Department of Otolaryngology/Head and Neck Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Address correspondence to: Scott Randell, 1117 Marsico Hall, Campus Box 7248, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7248, USA. Phone: 919.966.8093; Email: randell@med.unc.edu.

Authorship note: CAL and LH contributed equally to this work.

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1Marsico Lung Institute and Cystic Fibrosis Research Center,

2Department of Cell Biology and Physiology,

3Department of Microbiology and Immunology, and

4Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

5Icagen LLC, Durham, North Carolina, USA.

6Department of Otolaryngology/Head and Neck Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Address correspondence to: Scott Randell, 1117 Marsico Hall, Campus Box 7248, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7248, USA. Phone: 919.966.8093; Email: randell@med.unc.edu.

Authorship note: CAL and LH contributed equally to this work.

Find articles by Lewis, C. in: JCI | PubMed | Google Scholar

1Marsico Lung Institute and Cystic Fibrosis Research Center,

2Department of Cell Biology and Physiology,

3Department of Microbiology and Immunology, and

4Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

5Icagen LLC, Durham, North Carolina, USA.

6Department of Otolaryngology/Head and Neck Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Address correspondence to: Scott Randell, 1117 Marsico Hall, Campus Box 7248, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7248, USA. Phone: 919.966.8093; Email: randell@med.unc.edu.

Authorship note: CAL and LH contributed equally to this work.

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1Marsico Lung Institute and Cystic Fibrosis Research Center,

2Department of Cell Biology and Physiology,

3Department of Microbiology and Immunology, and

4Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

5Icagen LLC, Durham, North Carolina, USA.

6Department of Otolaryngology/Head and Neck Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Address correspondence to: Scott Randell, 1117 Marsico Hall, Campus Box 7248, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7248, USA. Phone: 919.966.8093; Email: randell@med.unc.edu.

Authorship note: CAL and LH contributed equally to this work.

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1Marsico Lung Institute and Cystic Fibrosis Research Center,

2Department of Cell Biology and Physiology,

3Department of Microbiology and Immunology, and

4Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

5Icagen LLC, Durham, North Carolina, USA.

6Department of Otolaryngology/Head and Neck Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Address correspondence to: Scott Randell, 1117 Marsico Hall, Campus Box 7248, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7248, USA. Phone: 919.966.8093; Email: randell@med.unc.edu.

Authorship note: CAL and LH contributed equally to this work.

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1Marsico Lung Institute and Cystic Fibrosis Research Center,

2Department of Cell Biology and Physiology,

3Department of Microbiology and Immunology, and

4Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

5Icagen LLC, Durham, North Carolina, USA.

6Department of Otolaryngology/Head and Neck Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Address correspondence to: Scott Randell, 1117 Marsico Hall, Campus Box 7248, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7248, USA. Phone: 919.966.8093; Email: randell@med.unc.edu.

Authorship note: CAL and LH contributed equally to this work.

Find articles by Mascenik, T. in: JCI | PubMed | Google Scholar

1Marsico Lung Institute and Cystic Fibrosis Research Center,

2Department of Cell Biology and Physiology,

3Department of Microbiology and Immunology, and

4Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

5Icagen LLC, Durham, North Carolina, USA.

6Department of Otolaryngology/Head and Neck Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Address correspondence to: Scott Randell, 1117 Marsico Hall, Campus Box 7248, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7248, USA. Phone: 919.966.8093; Email: randell@med.unc.edu.

Authorship note: CAL and LH contributed equally to this work.

Find articles by Dang, H. in: JCI | PubMed | Google Scholar |

1Marsico Lung Institute and Cystic Fibrosis Research Center,

2Department of Cell Biology and Physiology,

3Department of Microbiology and Immunology, and

4Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

5Icagen LLC, Durham, North Carolina, USA.

6Department of Otolaryngology/Head and Neck Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Address correspondence to: Scott Randell, 1117 Marsico Hall, Campus Box 7248, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7248, USA. Phone: 919.966.8093; Email: randell@med.unc.edu.

Authorship note: CAL and LH contributed equally to this work.

Find articles by Cholon, D. in: JCI | PubMed | Google Scholar |

1Marsico Lung Institute and Cystic Fibrosis Research Center,

2Department of Cell Biology and Physiology,

3Department of Microbiology and Immunology, and

4Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

5Icagen LLC, Durham, North Carolina, USA.

6Department of Otolaryngology/Head and Neck Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Address correspondence to: Scott Randell, 1117 Marsico Hall, Campus Box 7248, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7248, USA. Phone: 919.966.8093; Email: randell@med.unc.edu.

Authorship note: CAL and LH contributed equally to this work.

Find articles by Gentzsch, M. in: JCI | PubMed | Google Scholar |

1Marsico Lung Institute and Cystic Fibrosis Research Center,

2Department of Cell Biology and Physiology,

3Department of Microbiology and Immunology, and

4Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

5Icagen LLC, Durham, North Carolina, USA.

6Department of Otolaryngology/Head and Neck Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Address correspondence to: Scott Randell, 1117 Marsico Hall, Campus Box 7248, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7248, USA. Phone: 919.966.8093; Email: randell@med.unc.edu.

Authorship note: CAL and LH contributed equally to this work.

Find articles by Morton, L. in: JCI | PubMed | Google Scholar

1Marsico Lung Institute and Cystic Fibrosis Research Center,

2Department of Cell Biology and Physiology,

3Department of Microbiology and Immunology, and

4Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

5Icagen LLC, Durham, North Carolina, USA.

6Department of Otolaryngology/Head and Neck Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Address correspondence to: Scott Randell, 1117 Marsico Hall, Campus Box 7248, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7248, USA. Phone: 919.966.8093; Email: randell@med.unc.edu.

Authorship note: CAL and LH contributed equally to this work.

Find articles by Minges, J. in: JCI | PubMed | Google Scholar

1Marsico Lung Institute and Cystic Fibrosis Research Center,

2Department of Cell Biology and Physiology,

3Department of Microbiology and Immunology, and

4Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

5Icagen LLC, Durham, North Carolina, USA.

6Department of Otolaryngology/Head and Neck Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Address correspondence to: Scott Randell, 1117 Marsico Hall, Campus Box 7248, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7248, USA. Phone: 919.966.8093; Email: randell@med.unc.edu.

Authorship note: CAL and LH contributed equally to this work.

Find articles by Theile, J. in: JCI | PubMed | Google Scholar

1Marsico Lung Institute and Cystic Fibrosis Research Center,

2Department of Cell Biology and Physiology,

3Department of Microbiology and Immunology, and

4Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

5Icagen LLC, Durham, North Carolina, USA.

6Department of Otolaryngology/Head and Neck Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Address correspondence to: Scott Randell, 1117 Marsico Hall, Campus Box 7248, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7248, USA. Phone: 919.966.8093; Email: randell@med.unc.edu.

Authorship note: CAL and LH contributed equally to this work.

Find articles by Castle, N. in: JCI | PubMed | Google Scholar

1Marsico Lung Institute and Cystic Fibrosis Research Center,

2Department of Cell Biology and Physiology,

3Department of Microbiology and Immunology, and

4Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

5Icagen LLC, Durham, North Carolina, USA.

6Department of Otolaryngology/Head and Neck Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Address correspondence to: Scott Randell, 1117 Marsico Hall, Campus Box 7248, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7248, USA. Phone: 919.966.8093; Email: randell@med.unc.edu.

Authorship note: CAL and LH contributed equally to this work.

Find articles by Knowles, M. in: JCI | PubMed | Google Scholar

1Marsico Lung Institute and Cystic Fibrosis Research Center,

2Department of Cell Biology and Physiology,

3Department of Microbiology and Immunology, and

4Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

5Icagen LLC, Durham, North Carolina, USA.

6Department of Otolaryngology/Head and Neck Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Address correspondence to: Scott Randell, 1117 Marsico Hall, Campus Box 7248, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7248, USA. Phone: 919.966.8093; Email: randell@med.unc.edu.

Authorship note: CAL and LH contributed equally to this work.

Find articles by Kimple, A. in: JCI | PubMed | Google Scholar |

1Marsico Lung Institute and Cystic Fibrosis Research Center,

2Department of Cell Biology and Physiology,

3Department of Microbiology and Immunology, and

4Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

5Icagen LLC, Durham, North Carolina, USA.

6Department of Otolaryngology/Head and Neck Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Address correspondence to: Scott Randell, 1117 Marsico Hall, Campus Box 7248, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7248, USA. Phone: 919.966.8093; Email: randell@med.unc.edu.

Authorship note: CAL and LH contributed equally to this work.

Find articles by Randell, S. in: JCI | PubMed | Google Scholar |

Authorship note: CAL and LH contributed equally to this work.

Published July 28, 2022 - More info

Published in Volume 132, Issue 18 on September 15, 2022
J Clin Invest. 2022;132(18):e154571. https://doi.org/10.1172/JCI154571.
© 2022 Lee et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published July 28, 2022 - Version history
Received: September 20, 2021; Accepted: July 26, 2022 View PDF 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.

Graphical Abstractgraphical abstract Introduction

Cystic fibrosis (CF) is a life-limiting genetic disease affecting approximately 70,000 people worldwide (1). Severe pathology develops in the lungs, where absent or dysfunctional CF transmembrane regulator (CFTR) protein leads to the accumulation of thick airway mucus, impaired mucus transport, chronic infection and inflammation, and, eventually, bronchiectasis (2). Historically, treatments have been limited to symptom management. However, the 2012 US FDA approval of the first small-molecule CFTR modulator ivacaftor ushered in a new era of CF precision medicine (3). In contrast to previous treatment approaches, CFTR modulators treat the underlying cause of disease by directly acting on the CFTR protein to correct folding, trafficking, function, or stability. With additional CFTR modulator approvals (46) culminating in the 2019 approval of a triple-combination therapy, elexacaftor, tezacaftor, and ivacaftor (trade name Trikafta) (7), as many as 90% of individuals with CF are now eligible for an FDA-approved modulator therapy.

However, developing therapies for the remaining individuals with CF has proven challenging. This is in part because this patient group harbors a wide range of rare CFTR variants. Indeed, more than 1200 CFTR variants are carried by 5 or fewer individuals worldwide (8, 9). For these individuals, a well-powered clinical trial is not possible. Thus, to extend life-changing treatment to all people with CF, the approach for evaluating candidate therapies must evolve.

The FDA set the precedent for such a change in 2017, when they expanded the use of ivacaftor to patient populations harboring 1 of 23 relatively rare CFTR variants (10). Although drug label expansions are common, this instance was particularly groundbreaking because the FDA based their decision purely on in vitro data (11) rather than a clinical trial. This new paradigm relies on the fidelity of in vitro systems to accurately predict clinical responses.

Primary CF human bronchial epithelial cells (HBECs) obtained at the time of lung transplantation have served as the gold standard to assess CFTR rescue in vitro (1214). However, the supply of CF explant lungs is limited, particularly for rare CFTR variants. HBECs can also be obtained by bronchial brushing, but the procedure is invasive and yields low cell numbers (15). A readily available alternative to HBECs is human nasal epithelial cells (HNECs), which are increasingly being used as a model for the lower airways. HNECs can be obtained via nasal curettage, a nonsurgical and well-tolerated method (16). A direct comparison of paired HBEC and HNEC samples demonstrated that mature airway cell markers and CFTR activity with and without modulator treatment are preserved (15). However, like bronchial brushing, nasal curettage yields a limited supply of cells.

Previous work by our laboratory and others has shown that expression of mouse B cell–specific Moloney murine leukemia virus integration site 1 (Bmi-1) and human telomerase reverse transcriptase (hTERT) enables robust expansion of bronchial epithelial cells that can be differentiated and assayed for CFTR function for up to 15 passages (17, 18). Other groups have applied this method to nasopharyngeal biopsies (19), but, to our knowledge, this technique has not been used for nasal curettage samples. We hypothesized that Bmi-1 and hTERT expression would enhance HNEC growth properties, while maintaining their ability to differentiate into a polarized, pseudostratified epithelium. We propose that this method could be applied to rare-genotype HBECs and HNECs as they become available to create the cellular resources required for personalized medicine and drug discovery.

Here, we created 5 nasal and 3 bronchial Bmi-1/hTERT cell lines that recapitulated primary cell morphology and ion transport function for at least 15 passages. By examining cell lines with F508del/F508del and F508del/S492F genotypes and comparing them with the parent primary cells, we assessed the fidelity of CFTR modulator responses and the percentage of WT CFTR activity restored. We also compared the in vitro cell culture response to VX-445/VX-661 to the in vivo cell donors’ clinical responses to Trikafta.

We then used this approach to evaluate a potential modulator therapy for patients homozygous for W1282X, a nonsense mutation that generates a premature termination codon (PTC) in the CFTR transcript, predisposing CFTR mRNA to nonsense-mediated decay (NMD) and absent or truncated protein. Without a targetable protein, modulator therapies are ineffective, leaving these patients without treatment options. Recent studies illustrate that the cereblon (CRBN) E3 ubiquitin ligase modulator CC-90009 promotes PTC readthrough by knockdown of a key player in translation termination, the eukaryotic release factor 3a (eRF3a) (20). Using 1 nasal and 2 bronchial W1282X/W1282X cell lines, we tested the effects of CC-90009, again showing fidelity between cell lines and primary cells. Not only did we see robust CFTR rescue, but we also observed a dramatic reduction in epithelial sodium channel (ENaC) activity. We then assessed a second eRF3a degrader, SJ6986 (21), and obtained similar results, suggesting that eRF3a degradation represents a generalizable mechanism for the rescue of CFTR PTC variants. Finally, we tested CC-90009 and SJ6986 in a previously published but modified G542X/G542X bronchial cell line and observed dramatic synergy with the aminoglycoside G418 and robust CFTR functional rescue. We believe the cell lines and data generated here will facilitate the development of treatments for people with CF who currently lack an approved CFTR modulator therapy.

Results

Generation of Bmi-1/hTERT nasal and bronchial cell lines and culture optimization. Primary cell samples were transduced with a lentivirus containing mouse Bmi-1 and hTERT separated by the T2A self-cleaving peptide sequence (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/JCI154571DS1). By these methods, we generated 2 non-CF nasal, 3 CF nasal, and 2 CF bronchial cell lines (Table 1). We confirmed successful vector integration at passage 6 (P6) and P15 by hTERT activity and Bmi-1 protein expression (Supplemental Figure 1, B–F). Conditionally reprogrammed cell (CRC) culture improves HNEC growth capacity (2224). However, the CRC technique requires coculturing with irradiated or mitomycin-treated NIH3T3 feeder cells. We found that nasal cells could also be effectively expanded in EpiX media (Propagenix) as a feeder-free alternative. A comparison of a representative nasal cell line (UNCNN2T) and its parent cells in CRC and EpiX culture conditions (Supplemental Figure 1G) along with the growth properties of the other cell lines we generated can be found in the Supplemental Figure 2. Cell line differentiation was optimized by comparing UNC air-liquid interface (ALI) media (25), Vertex ALI media (26), and Pneumacult ALI media (STEMCELL Technologies). Pneumacult ALI reproducibly generated a mucociliary epithelial morphology (Supplemental Figure 3) and was used for the remainder of the study except where indicated.

Table 1

Demographics of the primary cell donors

Bmi-1/hTERT nasal cell lines model primary cell morphology and function. H&E and Alcian blue–periodic acid–Schiff (AB-PAS) staining of a representative non-CF nasal cell line (UNCNN2T) revealed a well-differentiated epithelium at P6 and P15 that was morphologically similar to the parent primary cells at P2 (Figure 1A). These results were confirmed by whole-mount immunostaining, which illustrated the presence of MUC5AC-producing goblet cells and α-tubulin+ ciliated cells (Figure 1, B–D). Measurements with a 24-channel transepithelial current clamp amplifier (TECC-24) device demonstrated that parent HNEC electrophysiology was also recapitulated by the UNCNN2T cell line at mid- and late-passage stages but with increased CFTR activity at P5 compared with that of parent cells (Figure 1, E–G). From this, we concluded that Bmi-1/hTERT nasal cell lines model primary HNEC morphology and function for at least 15 passages. Representative histology and whole-mount immunostaining of all other nasal cell lines are shown in Supplemental Figure 4.

Nasal cell lines model primary cell morphology and ion transport function.Figure 1

Nasal cell lines model primary cell morphology and ion transport function. (A) H&E and AB-PAS staining of UNCNN2T (non-CF) P2 parent cells and cell line at P6 and P15. Scale bar: 50 μm. (BD) Whole-mount immunostaining of UNCNN2T P2 parent cells (B) and cell line at P6 (C) and P15 (D). α-Tubulin (white), MUC5AC (green), phalloidin (F-actin, red), and Hoechst (nuclei, blue). Scale bar: 25 μm. (EG) TECC-24 measurements of UNCNN2T P2 parent cells and cell line at P5 and P15. (E) TECC-24 tracing representing 3–4 replicates. Acute addition of 6 μM benzamil (Benz), 10 μM FSK, and an inhibitor mixture (Inh mix) consisting of CFTRinh-172, GlyH-101, and bumetanide (each at 20 μM), is indicated by arrows. (F) Basal Ieq and change in Ieq (ΔIeq) in response to benzamil, FSK, and the inhibitor mixture. n = 3–4. (G) Baseline conductance values. n = 3–4. All data were analyzed using an ordinary linear model and are presented as the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.

Bmi-1/hTERT nasal cell lines predict the primary cell response to CFTR modulators. The goal of developing patient-derived cell lines is to generate a model in which the primary cell and ultimately the clinical response to CFTR-targeted therapies can be predicted. To test the ability of Bmi-1/hTERT nasal cell lines to predict CFTR modulator responses, we treated our F508del/F508del nasal cell line (UNCX4T) with CFTR corrector combinations or a vehicle control (DMSO). Here, and in all subsequent TECC-24 assays, CF cells were pretreated with test compounds and treated acutely with a CFTR potentiator (either genistein or VX-770 as indicated) following CFTR activation with forskolin (FSK). Thus, UNCX4T cells were treated with VX-445 and VX-661, the 2 correctors in Trikafta, or a recently described triple-corrector (3C) combination that combined VX-809 with 2 additional CFTR correctors, 3151 and 4172 (27). UNCX4T cells were then assayed for CFTR function using a TECC-24 device (Figure 2, A, C, and D). VX-445/VX-661 and 3C rescued 23.0% ± 1.4% and 19.5% ± 4.8% of WT CFTR function, respectively (mean ± SD), as determined by dividing the FSK response by the average non-CF nasal cell line response (i.e., 46.5 ± 5.0 μA/cm2) (Figure 1F and Supplemental Figure 2H). These data align with preclinical studies of VX-445/VX-661 and clinical observations of Trikafta in F508del homozygous populations (28). VX-445/VX-661 and 3C treatment also significantly rescued CFTR ion transport in the parent nasal cells compared with the DMSO control, rescuing 27.3% ± 5.1% and 26.7% ± 3.9% of WT CFTR function, respectively (mean ± SD) (Figure 2, B–D). These data suggest that the 3C combination might be as effective as Trikafta in rescuing F508del-CFTR and could represent a therapeutic candidate for those who cannot tolerate or do not respond to Trikafta treatment.

Nasal cell lines predict primary cell and clinical response to CFTR modulatFigure 2

Nasal cell lines predict primary cell and clinical response to CFTR modulators. (A and B) TECC-24 tracings of the UNCX4T nasal cell line (F508del/F508del) (A) and parent primary cells (B) treated with 0.1% DMSO and VX-445 and VX-661 (each at 5 μM), or a triple corrector combination (3C) containing VX-809/3151/4172 (each at 5 μM). Tracings are representative of 3–4 replicates. Acute addition of the potentiator 10 μM genistein is indicated by arrows. (C and D) ΔIeq of UNCX4T and parent cells in response to FSK (C) and the inhibitor mixture (D). n = 3–4. (E and F) TECC-24 tracings of the UNCX3T nasal cell line (F508del/S492F) (E) and parent primary cells (F) pretreated with DMSO, VX-445 and VX-661, or 3C. Tracings are representative of 3–4 replicates. (G and H) ΔIeq of UNCX3T and parent cells in response to FSK (G) and the inhibitor mixture (H). n = 3–4. (I) Change in the percentage of predicted FEV1 before and after Trikafta initiation in the UNCX4T donor (I) and the UNCX3T donor (J). Blue data points indicate FEV1 measured during SYMDEKO therapy, orange data points indicate FEV1 measured during Trikafta therapy, and red data points indicate FEV1 measured during a CF exacerbation. The treatment course for the UNCX4T and UNCX3T donors is indicated above the respective FEV1 plots and includes the timeline of i.v. antibiotics (green), XOLAIR for ABPA (dark blue), prednisone for ABPA (purple), antibiotics for treatment of Mycobacterium avium complex (MAC) (pink). (K) Change in weight in kilograms of the UNCX3T cell donor after Trikafta initiation. Gastrostomy tube use and subsequent removal are indicated by an orange bar. All data were analyzed using an ordinary linear model and are presented as the mean ± SD. Post hoc comparisons were performed using the general linear hypothesis test. **P < 0.01 and ***P < 0.001.

Next, we assessed a F508del/S492F compound heterozygous cell line (UNCX3T) for its response to VX-445/VX-661 and 3C (Figure 2, E, G, and H). UNCX3T responded well to VX-445/VX-661 treatment, recapitulating 22.3% ± 0.7% of WT CFTR function (mean ± SD). However, 3C treatment was less effective, producing 14.6% ± 1.5% of WT function (mean ± SD). These findings were mirrored in the UNCX3T parent cells (Figure 2, F–H). Because 3C treatment produced a lower CFTR functional response in UNCX3T, which carries only 1 copy of F508del, we posit that this modulator combination does not rescue S492F-CFTR as effectively as F508del-CFTR. Even so, the response to 3C fell well within the therapeutic window (i.e., 10% of WT CFTR function over baseline) (29) and could serve as an alternative therapy. From these studies, we concluded that Bmi-1/hTERT nasal cell lines generated from CF donors can be used to accurately predict the primary cell response to FDA-approved CFTR modulators and those in preclinical development.

Nasal cell line functional rescue correlates with the clinical response to Trikafta in 2 patients with CF. All nasal curettage samples were obtained prior to the 2019 FDA approval of Trikafta. At the time of cell collection, the UNCX4T cell donor (F508del/F508del) was prescribed tezacaftor/ivacaftor (trade name SYMDEKO), whereas the UNCX3T cell donor (F508del/S492F) was not eligible for CFTR modulators. After nasal cell harvest and cell line generation, both donors became eligible for Trikafta and initiated treatment. Trikafta therapy was highly effective in these individuals, with an 11% and 22% increase in the percentage of predicted forced expired volume in 1 second (FEV1) over a 6-month baseline in the UNCX4T and UNCX3T donors, respectively (Figure 2, I and J). For the UNCX3T donor, Trikafta therapy promoted other significant changes in health, including a reduced frequency of pulmonary exacerbations and need for i.v. antibiotics, cessation of XOLAIR treatment for allergic bronchopulmonary aspergillosis (ABPA), and gastrostomy tube removal following improved weight gain and retention (Figure 2, J and K). Overall, the robust functional response to Trikafta that was observed in the UNCX4T and UNCX3T nasal cell lines in vitro correlated with the cell donors’ positive clinical response to therapy.

W1282X-CFTR is rescued by the CRBN modulator CC-90009. Current therapies are not effective at rescuing the truncated protein generated by the W1282X-CFTR variant. One proposed treatment strategy is to promote ribosomal readthrough of the PTC to generate full-length protein (30, 31). Low levels of readthrough can be accomplished with high concentrations of aminoglycosides in overexpression cell lines (32). However, clinical readthrough agents are largely ineffective (33, 34), probably because of NMD, a surveillance pathway by which the cell detects and eliminates PTC-containing mRNA transcripts (35). Indeed, a recent study found substantial degradation of the CFTR transcript in an individual homozygous for the W1282X variant, with the mutated CFTR mRNA expressed at only 2.1% of WT levels (36). Thus, many groups have hypothesized that effective treatment of nonsense mutations will also require NMD inhibition (31, 36, 37). Studies in which NMD is bypassed by expressing intron-less cDNA copies of W1282X-CFTR have demonstrated that the truncated protein exhibits defective cellular trafficking and gating that can be augmented by CFTR modulators (38, 39). Yet in primary cells, CFTR modulators alone do not rescue function (30). We therefore hypothesized that effective rescue of W1282X-CFTR would require a combination of therapeutic approaches to (a) promote PTC readthrough, (b) inhibit NMD, and (c) modulate the resulting CFTR protein.

Recently, a class of CRBN E3 ligase modulators have been shown to significantly improve PTC readthrough by aminoglycoside compounds (20). One of these agents, CC-90009, is currently under investigation for the treatment of relapsed and refractory acute myeloid leukemia (AML) in a phase I clinical trial (NCT02848001). CC-90009 mediates the interaction between CRBN and eRF3a, also known as the G1 to S phase transition 1 (GSPT1) protein, which functions as a key player in stop codon recognition and translation termination. Upon interaction with CRBN, eRF3a is ubiquitinated and targeted for proteasomal degradation. Thus, we hypothesized that CC-90009 would further enhance therapeutic readthrough and rescue of W1282X-CFTR.

We optimized the CC-90009 dose by treating primary HBECs with escalating concentrations from 0.01 to 10 μM and probing for eRF3a protein knockdown (Supplemental Figure 5A). Knockdown of 85% was achieved with CC-90009 concentrations of 0.1 μM or higher. We then assessed cytotoxicity by measuring lactate dehydrogenase (LDH) release and loss of transepithelial resistance in ALI cultures treated with CC-90009 doses ranging from 0.1 to 1 μM (Supplemental Figure 5, B–D). Cytotoxicity was not observed with 0.1 μM CC-90009, although it was seen at higher doses. Cell morphology was also not altered by 0.1 μM CC-90009 treatment (Supplemental Figure 5E). From this, we concluded that 0.1 μM CC-90009 effectively reduced eRF3a protein expression without causing cytotoxicity or aberrant changes in cell morphology.

Having established a safe dose, we then treated a W1282X/W1282X nasal cell line (UNCX2T) with combinations of CC-90009, an inhibitor of NMD (Smg1i), an aminoglycoside (G418), and a CFTR corrector (VX-809) and compared the results with a vehicle control (DMSO) and single drug controls (Figure 3, A–C). CFTR function (i.e., FSK and CFTRinh-172 response) was undetectable in vehicle-treated cells or cells treated with Smg1i or G418 alone. However, all combinations that included CC-90009 resulted in substantial CFTR rescue. Unexpectedly, the CC-90009 single drug control rescued 19.1% ± 3.5% of WT CFTR function, indicating that CC-90009 was primarily responsible for the rescue seen in the tested combinations. Unlike previous reports (20), we did not observe synergy between G418 and CC-90009 to rescue W1282X-CFTR. The sufficiency of CC-90009 to rescue CFTR was confirmed in a panel of 3 W1282X/W1282X cell lines (UNCX2T, UNCCF9T, and UNCCF10T) and in the parent primary cells (Figure 3, D–G), where CFTR was rescued to 21.7% ± 5.0% and 19.5% ± 6.0% of WT CFTR function, respectively. From this, we concluded that CC-90009 could function as a single agent to rescue W1282X-CFTR to approximately 20% of WT function.

The eRF3a degrader CC-90009 rescues W1282X-CFTR in a panel of cell lines anFigure 3

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

CC-90009 diminishes ENaC function and expression. ENaC plays a vital role in the lungs, balancing CFTR-mediated fluid secretion by regulating sodium absorption across the airway epithelium. Upon treatment with CC-90009, we noted a striking and unexpected decrease in ENaC function, indicated by the response to benzamil, a potent ENaC inhibitor (Figure 3H). Indeed, CC-90009 diminished the benzamil response by 93.4% ± 3.8% and 84.3% ± 6.1

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