(686) High-Throughput Functional Assay using Patient-Derived Organoids allows Drug Repurposing for Cystic Fibrosis

Background: Recent therapies enable effective restoration of CFTR function of the most prevalent F508del CFTR mutation and shift the clinical unmet need largely towards people with rare CFTR mutations. Approximately 10% of the worldwide CF population carry premature termination codons (PTC) or nonsense mutations, resulting in truncated CFTR protein with severe loss-of-function. Previous research identified compounds with read-through (RT) activity that result in introduction of an amino acid at the PTC site. At present-day, ELX-02 is the only RT-compound currently in a clinical trial (phase II) for CF patients with at least one G542X allele (NCT04135495). This underlines the need to continue the search for novel CF therapeutics. Drug repurposing of FDA approved compounds could potentially aid in this. A prerequisite for drug repurposing, is that the exploited assay is robust as well as clinically relevant. Forskolin induced swelling (FIS) measurements in patient-derived intestinal organoids (PDIOs) allow characterization of CFTR function, and have been described to associate with clinical features of CF thereby enabling drug repurposing in a personalized setting¹.

Methods: We upscaled the previously described FIS assay² to a 384-wells format by adapting several aspects of the protocol. We validated assay robustness in several ways, after which we characterized toxicity of 1443 FDA-approved compounds. The non-toxic compounds were screened for potential to elevate CFTR-mediated fluid secretion in 4 PDIO lines homozygously expressing W1282X CFTR, one of the most prevalent PTC mutations. CFTR modulators lumacaftor/ivacaftor (Orkambi) were added to all conditions, to elevate baseline CFTR function and increase the chance of hit detection.

Results: We show that the 384-wells FIS-assay is reproducible, has a large wide dynamic range and yields similar responses as obtained in the conventional 96-wells based set-up. Hereafter, compound-induced toxicity was quantified and 43 compounds were excluded for future experiments. Results of the drug repurposing FIS assay identified 19 potential inducers of organoid fluid secretion. The top 5 hits were further characterized in a secondary screen allowing identification of members of the statin family as compounds that can rescue CFTR function. Statins are commonly used medicines to lower blood pressure by inhibiting HMG CoA reductase and have not elaborately been described to affect CFTR function³. We are currently performing experiments to further clarify genotype specificity and potential mode-of-action of statin-mediated increase of CFTR function.

Conclusions: Our study exemplifies the feasibility of large-scale compound screening with a functional read-out using patient derived organoids. The pipeline of this study is summarized in Fig. 1. Furthermore we show a not earlier described interaction between CFTR and members of the statin family in patient-derived organoids harbouring PTC mutations.

Acknowledgements: This work was supported by Emily’s Entourage and Health Holland (TKI).

References: [1] Berkers, Gitte, et al. "Rectal organoids enable personalized treatment of cystic fibrosis." Cell reports 26.7 (2019): 1701-1708.

[2] Vonk, Annelotte M., et al. "Protocol for application, standardization and validation of the forskolin-induced swelling assay in cystic fibrosis human colon organoids." STAR protocols 1.1 (2020): 100019.

[3] Vaughan, Carl J., Michael B. Murphy, and Brendan M. Buckley. "Statins do more than just lower cholesterol." The Lancet 348.9034 (1996): 1079-1082.