(598) Alternate start site M265 allows 5’ nonsense variants to escape NMD so that readthrough and modulators can restore CFTR function

Background: Variants that do not produce CFTR protein are challenging to therapeutically target. Nearly half of disease-causing variants in CFTR are predicted to introduce a premature termination codon (PTC), and these transcripts are targeted for degradation by nonsense mediated mRNA decay (NMD), resulting in unstable CFTR mRNA and lack of CFTR protein. However, nonsense variants in the 5’ end of CFTR escape NMD resulting in stabilized CFTR transcript. In this study, we evaluated whether initiation of translation at start sites downstream of a 5’ nonsense variant prevents NMD (due to removal of exon junction complexes) and whether the stable CFTR transcript can be targeted by readthrough agents to introduce an alternate amino acid at the PTC, allowing for production of a full-length CFTR protein.

Methods: Expression minigenes (EMGs) containing select abridged or full-length CFTR intronic sequences were generated bearing variants E60X, L88X, and Y122X, and were transiently expressed in HEK293 cells. Alternative translation initiation methionine at codons 150, 152 and 265 were changed to valine by site-directed mutagenesis. Immunoblotting of cell lysates was used to evaluate CFTR protein processing. Primary human nasal epithelial (NE) cells from individuals carrying L88X were conditionally reprogrammed and grown at air liquid interface. Transcript stability was analyzed by reverse transcription (RT)-PCR. Cells were treated with readthrough agent G418 (100-400µM) or ELX-02 (100-200µM) and CFTR correctors (Elexa and Teza, 3µM). CFTR function was measured by mounting cells on Ussing chambers to record short-circuit current (Isc) values by acute addition of agonists (forskolin and ivacaftor), and antagonists (Inh-172).

Results: Immunoblotting showed that untreated HEK293 cells expressing E60X and L88X CFTR-EMG generated two CFTR-specific core glycosylated products, consistent with downstream translation initiation. Mutation of methionine at positions 150 and 152 did not result in changes in CFTR protein processing in L88X HEK293s. However, mutation of M265 resulted in loss of visible CFTR protein in cells expressing E60X, L88X, and Y122X CFTR­-EMGs and resulted in no visible change in WT-CFTR­-EMG cells, indicating that downstream translation initiation is occurring at M265.
RT-PCR of WT/L88X NE cells revealed that L88X mRNA is stable and comparable to WT levels. In functional assessments, WT/WT NE cells generated CFTR function of 13.1±1.5µA/cm² (n=12). Untreated L88X/F508del NE cells generated 6.2±0.8% of WT function. Triple combination alone restored function to 83±10.9% of WT. Previously, treatment with G418 and triple combination in CFBE cells stably expressing L88X-EMG resulted in robust increase in current, up to 57% of WT. Here, treatment with G418 and triple combination in L88X/F508del NE cells did not result in additional recovery of function. However, treatment with ELX-02 and triple combination resulted in robust recovery of CFTR function to 122.8±14.1% of WT. Notably, G418 resulted in significant decrease in amiloride response which was not observed with ELX-02, indicating that recovery of CFTR function with ELX-02 is not related to its action on ENaC activity.

Conclusions: Downstream translation initiation of 5’ nonsense variants occurs at M265, resulting in stable CFTR transcript. Thus, triple combination therapy combined with readthrough may be a suitable treatment option for 5’ CFTR variants that evade NMD.

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