FDA-approved drug screening in patient-derived organoids demonstrates potential of drug repurposing for rare cystic fibrosis genotypes.

BACKGROUND: Preclinical cell-based assays that recapitulate human disease play an important role in drug repurposing. We previously developed a functional forskolin induced swelling (FIS) assay using patient-derived intestinal organoids (PDIOs), allowing functional characterization of CFTR, the gene mutated in people with cystic fibrosis (pwCF). CFTR function-increasing pharmacotherapies have revolutionized treatment for approximately 85% of people with CF who carry the most prevalent F508del-CFTR mutation, but a large unmet need remains to identify new treatments for all pwCF. METHODS: We used 76 PDIOs not homozygous for F508del-CFTR to test the efficacy of 1400 FDA-approved drugs on improving CFTR function, as measured in FIS assays. The most promising hits were verified in a secondary FIS screen. Based on the results of this secondary screen, we further investigated CFTR elevating function of PDE4 inhibitors and currently existing CFTR modulators. RESULTS: In the primary screen, 30 hits were characterized that elevated CFTR function. In the secondary validation screen, 19 hits were confirmed and categorized in three main drug families: CFTR modulators, PDE4 inhibitors and tyrosine kinase inhibitors. We show that PDE4 inhibitors are potent CFTR function inducers in PDIOs where residual CFTR function is either present, or created by additional compound exposure. Additionally, upon CFTR modulator treatment we show rescue of CF genotypes that are currently not eligible for this therapy. CONCLUSION: This study exemplifies the feasibility of high-throughput compound screening using PDIOs. We show the potential of repurposing drugs for pwCF carrying non-F508del genotypes that are currently not eligible for therapies. ONE-SENTENCE SUMMARY: We screened 1400 FDA-approved drugs in CF patient-derived intestinal organoids using the previously established functional FIS assay, and show the potential of repurposing PDE4 inhibitors and CFTR modulators for rare CF genotypes.

Functional Restoration of CFTR Nonsense Mutations in Intestinal Organoids.

BACKGROUND: Pharmacotherapies for people with cystic fibrosis (pwCF) who have premature termination codons (PTCs) in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are under development. Thus far, clinical studies focused on compounds that induce translational readthrough (RT) at the mRNA PTC location. Recent studies using primary airway cells showed that PTC functional restoration can be achieved through combining compounds with multiple mode-of-actions. Here, we assessed induction of CFTR function in PTC-containing intestinal organoids using compounds targeting RT, nonsense mRNA mediated decay (NMD) and CFTR protein modulation. METHODS: Rescue of PTC CFTR protein was assessed by forskolin-induced swelling of 12 intestinal organoid cultures carrying distinct PTC mutations. Effects of compounds on mRNA CFTR level was assessed by RT-qPCRs. RESULTS: Whilst response varied between donors, significant rescue of CFTR function was achieved for most donors with the quintuple combination of a commercially available pharmacological equivalent of the RT compound (ELX-02-disulfate or ELX-02ds), NMD inhibitor SMG1i, correctors VX-445 and VX-661 and potentiator VX-770. The quintuple combination of pharmacotherapies reached swelling quantities higher than the mean swelling of three VX-809/VX-770-rescued F508del/F508del organoid cultures, indicating level of rescue is of clinical relevance as VX-770/VX-809-mediated F508del/F508del rescue in organoids correlate with substantial improvement of clinical outcome. CONCLUSIONS: Whilst variation in efficacy was observed between genotypes as well as within genotypes, the data suggests that strong pharmacological rescue of PTC requires a combination of drugs that target RT, NMD and protein function.

Intestinal organoids for Cystic Fibrosis research.

Significant progress has been made in the development of CFTR modulator therapy; however, current CFTR modulator therapies are only available for a minority of the CF-patient population. Additionally, heterogeneity in in vivo modulator response has been reported among individuals carrying homozygous F508del-CFTR, adding to the desire for an optimal prediction of response-to-therapy on an individual level. In the last decade, a lot of progress has been made in the development of primary cell cultures into 3D patient-derived disease models. The advantage of these models is that the endogenous CFTR function is affected by the patient's mutation as well as other genetic or environmental factors. In this review we focus on intestinal organoids as in vitro model for CF, enabling for CF disease classification, drug development and treatment optimization in a personalized manner, taking into account rare CFTR mutations and clinical heterogeneity among individuals with CF.

Microsatellite diversity of the Nordic type of goats in relation to breed conservation: how relevant is pure ancestry?

In the last decades, several endangered breeds of livestock species have been re-established effectively. However, the successful revival of the Dutch and Danish Landrace goats involved crossing with exotic breeds and the ancestry of the current populations is therefore not clear. We have generated genotypes for 27 FAO-recommended microsatellites of these landraces and three phenotypically similar Nordic-type landraces and compared these breeds with central European, Mediterranean and south-west Asian goats. We found decreasing levels of genetic diversity with increasing distance from the south-west Asian domestication site with a south-east-to-north-west cline that is clearly steeper than the Mediterranean east-to-west cline. In terms of genetic diversity, the Dutch Landrace comes next to the isolated Icelandic breed, which has an extremely low diversity. The Norwegian coastal goat and the Finnish and Icelandic landraces are clearly related. It appears that by a combination of mixed origin and a population bottleneck, the Dutch and Danish Land-races are separated from the other breeds. However, the current Dutch and Danish populations with the multicoloured and long-horned appearance effectively substitute for the original breed, illustrating that for conservation of cultural heritage, the phenotype of a breed is more relevant than pure ancestry and the genetic diversity of the original breed. More in general, we propose that for conservation, the retention of genetic diversity of an original breed and of the visual phenotype by which the breed is recognized and defined needs to be considered separately.

Practical measurement of affordability: an application to medicines.

OBJECTIVE: To develop two practical methods for measuring the affordability of medicines in developing countries. METHODS: The proposed methods--catastrophic and impoverishment methods--rely on easily accessible aggregated expenditure data and take into account a country's income distribution and absolute level of income. The catastrophic method quantifies the proportion of the population whose resources would be catastrophically reduced by spending on a given medicine; the impoverishment method estimates the proportion of the population that would be pushed below the poverty line by procuring a given medicine. These methods are illustrated by calculating the affordability of glibenclamide, an antidiabetic drug, in India and Indonesia. The results were validated by comparing them with the results obtained by using household micro data for India and Indonesia. FINDINGS: When accurate aggregate data are available, the proposed methods offer a practical way to obtain informative and accurate estimates of affordability. Their results are very similar to those obtained with household micro data analysis and are easily compared across countries. CONCLUSION: The catastrophic and impoverishment methods, based on macro data, can provide a suitable estimate of medicine affordability when the household level micro data needed to carry out more sophisticated studies are not available. Their usefulness depends on the availability of accurate aggregated data.