Suen, S. | Search bioRxiv

bioRxiv2026

A functional comparison of readthrough agent ELX-02 across a wide range of nonsense CFTR variants

BackgroundNonsense variants in CFTR account for ~10% of cystic fibrosis (CF) variants and cannot be treated with approved CFTR modulators. Translational readthrough agents such as ELX-02 offer a potential therapeutic strategy, but clinical trials evaluated mainly in G542X CFTR nonsense variant and underlined limited efficacy. This study aimed to evaluate ELX-02-mediated CFTR rescue across a broad range of nonsense variants using patient-derived intestinal organoids (PDIOs) to define variant-specific determinants of readthrough efficacy and assess its potential across a genetically diverse CF population. MethodThe ex vivo response to ELX-02 was assessed in 206 PDIOs carrying heterogeneous nonsense variants. CFTR function was quantified using forskolin-induced swelling (FIS) assay after 48-hour exposure to ELX-02. Responses were analysed by genotype and stop codon identity, with secondary validation performed in a selected subset of PDIOs (n = 60). ResultsELX-02-mediated CFTR rescue varied markedly, ranging from responses approaching those observed with approved CFTR modulators (LUM/IVA) to responses at or below detection limit. Overall, maximal responses were modest and at the lower end of the functional range for CFTR modulators. Rescue was dose-dependent and higher in PDIOs carrying two nonsense variants when compared with PDIOs carrying a single nonsense variant combined with a residual or minimal function variant. Nonsense variants in nucleotide-binding domain 1, including G542X, S466X, G550X and R553X, showed relatively higher responsiveness. ConclusionELX-02 induces limited and highly heterogeneous CFTR rescue across nonsense variants. PDIO-based functional screening provides a framework to guide patient selection and stratification for future readthrough therapy trials.

molecular biology↗

bioRxiv2022

High-Throughput Functional Assay in Cystic Fibrosis Patient-Derived Organoids Allows Drug Repurposing

Cystic fibrosis (CF) is a rare hereditary disease caused by mutations in the CFTR gene. Recent therapies enable effective restoration of CFTR function of the most common F508del CFTR mutation. This shifts the unmet clinical need towards people with rare CFTR mutations such as nonsense mutations, of which G542X and W1282X are most prevalent. CFTR function measurements in patient-derived cell-based assays played a critical role in preclinical drug development for CF and may play an important role to identify new drugs for people with rare CFTR mutations. Here, we miniaturized the previously described forskolin induced swelling (FIS) assay in intestinal organoids from a 96-wells to a 384-wells plate screening format. Using this novel assay, we tested CFTR increasing potential of a 1400-compound FDA-approved drug library in organoids from donors with W1282X/W1282X CFTR nonsense mutations. The 384-wells FIS-assay demonstrated uniformity and robustness based on CV and Z-factor calculations. In the primary screen, the top 5 compound combinations that increased CFTR function all contained at least one statin. In the secondary screen, we indeed verified that four out of the five statins, Mevastatin; Lovastatin; Simvastatin and Fluvastatin increased CFTR function when combined with CFTR modulators. Statin-induced CFTR rescue was W1282X specific, as increased CFTR function was not shown for patient-derived organoids harbouring R334W/R334W and F508del/F508del mutations. Future studies should focus on elucidating genotype specificity and mode-of-action of statins into more detail. This study exemplifies proof-of-principle of large-scale compound screening in a functional assay using patient derived organoids. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/500147v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@fccbfdorg.highwire.dtl.DTLVardef@b459ceorg.highwire.dtl.DTLVardef@7414fborg.highwire.dtl.DTLVardef@10d9c35_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗