(613) Understanding synergy in nonsense suppression therapy for CFTR nonsense mutations

Background: 10% of cystic fibrosis (CF) patients carry a nonsense mutation, which generates a premature termination codon (PTC) in the CFTR mRNA. A PTC terminates translation before a full-length protein is made, producing negligible CFTR expression/function. CFTR modulators, which target CFTR protein, alleviate CF in the 90% of patients who retain partial CFTR expression. However, modulators are ineffective in CF patients who carry nonsense mutations due to the near absence of CFTR. One approach to treat CF patients with nonsense mutations is nonsense suppression therapy. This approach uses small molecules to suppress translation termination at PTCs. This approach works by a readthrough mechanism where an amino acid is inserted into the nascent peptide at the site of the PTC, allowing translation to continue downstream of the PTC in the correct reading frame to rescue full-length, functional protein. Low efficacy of current readthrough agents led us to perform a search for more effective readthrough compounds.

Methods: To identify new readthrough compounds, we performed a high throughput screen of >700,000 low molecular weight compounds using Fischer rat thyroid (FRT) cells stably expressing a NanoLuc-based readthrough reporter for compounds that effectively suppress termination at PTCs without toxicity. Hit compounds were further validated for readthrough using a variety of mechanism-based reporters. The most promising compounds were then tested for their ability to restore CFTR expression and function in a variety of cell types, including gene-edited, human bronchial epithelia (16HBE14o-) and primary HBE cells obtained from CF patients.

Results: From this screen, we identified a novel class of readthrough agent that depletes the termination factor eRF1 by targeting it for proteasomal degradation. eRF1 degraders alone modestly suppressed CFTR PTCs; however, when combined with aminoglycosides such as G418 or ELX-02, synergistic increases in readthrough were observed. By examining eRF1 degraders in combination with other classes of readthrough agents to find those that best synergize, we discovered that PTC-containing mRNA abundance progressively increases as readthrough is enhanced, suggesting that mRNA abundance plays a key role in generating readthrough synergy, and the there is a direct relationship between the level of readthrough and the amount of NMD inhibition.

Conclusions: Combining different functional classes of readthrough compounds can lead to synergistic increases in readthrough, restore a significant amount of mRNA, thereby resulting in the restoration of a significant level of CFTR function. The development of specific combinations of readthrough compounds that act synergistically is likely to generate treatments that can rescue clinically significant levels of CFTR function.

Acknowledgements: This work was supported by the Cystic Fibrosis Foundation subaward 858345.

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