(691) Synthetic aminoglycoside ELX-02 induces readthrough of CFTR-G550X producing super-functional protein that can be further enhanced by CFTR correctors

Background: No approved therapies exist for premature termination codons (PTC) mutations, which present in ~11% of people with CF. The novel aminoglycoside derivative ELX-02 promotes readthrough of PTC-bearing CFTR transcripts by recruiting near-cognate aminoacyl tRNAs to the PTCs and shows reduced toxicity compared to other aminoglycosides such as G418. The amino acids inserted at PTCs, which can affect the processing and function of the resulting CFTR, are largely context-dependent. We discovered G550X may be particularly responsive to readthrough agents in intestinal organoids derived from six G550X donors, suggesting it may be unique in its response characteristics.

Methods: A rare nonsense mutation in CFTR (G550X) was compared to a more common nonsense mutation (G542X) upon G418 or ELX-02 treatment. First, a reporter construct containing the G550X context (TGFP-G550X-HA-6XHis) in HEK cells was used to identify the amino acids that are inserted at the PTC of G550X by mass-spec analysis. Second, isogenic FRT cells expressing the G550 CFTR variants were generated and subject to Isc and patch-clamp studies. Third, readthrough and CFTR function by transepithelial chloride conductance (TECC) were measured in response to readthrough drugs and CFTR modulators.

Results: Intestinal organoids with G550X had a robust response to ELX-02 in vitro. HEK cells with a His-tagged readthrough reporter construct containing the G550X CFTR context were treated with G418 or ELX-02. Analysis of the readthrough protein subjected to mass spectrometry revealed tryptophan (W) as the sole amino acid inserted in the G550X position, dissimilar to G542X which exhibited three near-cognate amino acid possibilities. FRT cells expressing G550W CFTR exhibited significantly increased forskolin activated Cl^- conductance compared to WT-CFTR (53.1 ± 6.9, vs. 26.0 ± 2.3 mS/cm², p< 0.0001). In addition, patch-clamp studies showed that G550W CFTR channel had increased PKA sensitivity (EC50: 19.6 vs. 49.5 nM, p< 0.01) and open probability (0.41 ± 0.05 vs. 0.22 ± 0.04, p< 0.01) compared to WT CFTR. CFTR meditated Cl^- conductance was evident when FRT cells expressing G550X or G542X CFTR were treated with ELX-02 or G418, but improvement of CFTR function was greater with G550X than G542X (ELX-02: 4.57 ± 0.60, vs. 0.38 ± 0.02 and G418: 8.08 ± 1.29, vs. 0.67 ± 0.04; P< 0.0001). Conductance was further augmented by combined treatment with CFTR correctors (elexacaftor and tezacaftor (ET)) and potentiators (ivacaftor), achieving ~20% (ELX-02+ET) and ~40% (G418+ET) of WT conductance in isogenic FRT comparators. Western blotting was consistent with functional results.

Conclusions: Compared to the more common G542X PTC mutation, G550X has a greater response to aminoglycosides due to gain-of-function properties of the CFTR readthrough product. This is explained by the insertion of tryptophan in the G550 position augments PKA sensitivity and channel gating. In combination with the clinically approved CFTR correctors, the function of G550X CFTR can be improved to ~20%-40% of the wild-type level. G550X may be particularly sensitive target for translational readthrough therapy.

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