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

Abstract

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.

FDA-Approved Drug Screening in Patient-Derived Organoids Demonstrates Potential of Drug Repurposing for Rare Cystic Fibrosis Genotypes

“…The procedure to biobank and culture PDIOs for screening was robust. Previous optimization of several steps in 96-wells FIS assay 4 , allowed us to practically perform the FIS assay in a 384-wells format 9 .In the context of highthroughput screenings, most efforts have been made in the cancer-field. Whilst patient-derived organoid screening has received much attention, many studies in this context are lower-throughput screenings, in which read-outs are often centered around viability [17][18][19] .…”

“…384-wells FIS-assays were performed according to previously described protocols 4,5 , with minor adaptations allowing a 384-wells screening setting as summarized in Table 3 9 . PDIOs of 76 different donors were seeded in 25% matrigel on two 384-wells plates/donor (7 µL/well).…”

“…Other prerequisites for drug repurposing are that the exploited assay is high-throughput and robust. We recently succeeded in establishing a high-throughput screening version of the FIS assay, allowing testing of compounds that directly or indirectly influence CFTR function in a high-throughput manner on CF patient-derived material 9 . Using this miniaturized FIS assay, we screened 76 non-homozygous F508del PDIOs, aiming to identify CFTR function enhancing drugs in a 1400-compound FDA-approved drug library.…”

“…Other prerequisites for drug repurposing are that the exploited assay is high-throughput and robust. We recently succeeded in establishing a high-throughput screening version of the FIS assay, allowing testing of compounds that directly or indirectly influence CFTR function in a high-throughput manner on CF patient-derived material 9 .…”

Drug repurposing for Cystic Fibrosis: identification of drugs that induce CFTR-independent fluid secretion in nasal organoids

“…In this study, we performed a screening assay in CF nasal organoids, with the aim to repurpose FDAapproved drugs that stimulate CFTR-independent fluid secretion. Screening assays in 384-wells plate format using airway organoids have been previously been described by others [23][24][25] and our protocol was based on a 384-wells screening assay for CFTR-modulating drugs in CF intestinal organoids [26]. However, to our knowledge, this is the first medium-throughput 384-wells screening assay using nasal airway organoids that are cultured from minimal invasive nasal brushings of pwCF.…”