(614) Sensitized high throughput screening for translational readthrough promoters in the context of the native CFTR R1162X gene

Background: About 10% of people with cystic fibrosis (PwCF) carry at least one CFTR allele with a premature termination codon (PTC) mutation. Approximately 170 CFTR PTC variants have been reported. Developing effective therapies that target the molecular defects of these variants is an unmet need for PwCF carrying CFTR nonsense mutations.

Recent in vitro data from combinations of aminoglycosides with degraders of translation termination or peptide release factors are encouraging and a promising first step towards developing effective, readthrough promoting combination therapies [1]. Building on this concept, we utilized the cereblon E3 ligase inhibitor CC-90009 that induces proteasomal degradation of eRF3a, to sensitize a high throughput screening assay with the goal to identify novel readthrough modulators that may synergize with an eRF3 degrader mechanism.

Methods: Utilizing CRISPR/Cas9 editing capabilities, we generated multiple clonal cell lines expressing PTC variants (Y122X, G542X, R1162X, and W1282X) from the native CFTR gene of 16HBE14o^- cells. We also developed biochemical assays (ELISA and Western blotting) to evaluate biochemical readthrough of native PTC CFTR constructs and performed functional measurements in the electrophysiological (TECC-24) equivalent current (I_eq) assay.

Results: We determined the 16HBEge R1162X CFTR cell line to be the most sensitive cell model for readthrough when treated with aminoglycosides and eRF degraders, achieving 20% of WT CFTR protein expression levels for ELX-02/CC-90009 combination treatments.

Using gene-editing, we then generated a 16HBEge R1162X CFTR cell line with a strong (EF1a) promoter in the 5’ UTR and a C-terminally fused NanoLuc reporter to enable screens for readthrough active molecules in a high throughput fashion. Here, we will present our findings from screening of a small library of 368 antibiotics in the presence of eRF3a degrader CC-90009. The library contains antibiotics with multiple mechanisms of action. Our most efficacious hit compound (tedizolid) is a ribosomal modulator of the oxazolidinone class. Currently ongoing is the screen of the 12,160 compound ReFrame library (Calibr, LaJolla, CA). 13 HTS hits (0.1%) were more efficacious than 0.2 mM G418. Further HTS and hit validation are in progress.

Conclusions: Together, our data strongly support that the combination of eRF3a degraders and ribosomal modulators is an efficient approach to pharmacological readthrough of CFTR PTCs. In addition, we demonstrate that our high throughput screen in a cell model that accounts for all relevant biology in the context of premature translation termination (CFTR pre-mRNA splicing, NMD) can be a viable approach to identify additional agents with efficient readthrough promoting activity.

Acknowledgements: We thank E. Wong (CFFT Lab), Drs. D.M. Bedwell and S.M. Rowe (UAB) for discussions and suggestions.

References: [1] Sharma J, Du M, Wong, E., Mutyam V, Li Y, Chen J, Wangen J, Thrasher K, Fu L, Peng N, Tang L, Liu K, Mathew B, Bostwick RJ, Augelli-Szafran CE, Bihler H, Liang F, Mahiou J, Saltz J, Rab A, Hong J, Sorscher EJ, Mendenhall EM, Coppola CJ, Keeling KM, Green R, Mense M, Suto MJ, Rowe SM, Bedwell DM. A small molecule that induces translational readthrough of CFTR nonsense mutations by eRF1 depletion. Nat Commun. 2021; 12:4358. 10.1038/s41467-021-24575-x