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.

Targeted locus amplification reveals heterogeneity between and within CFTR genotypes and association with CFTR function in patient-derived intestinal organoids.

BACKGROUND: Cystic fibrosis (CF) disease severity can be highly variable, even between people with CF (pwCF) with similar genotypes. Here we use patient-derived intestinal organoids to study the influence of genetic variation within the cystic fibrosis transmembrane conductance regulator (CFTR) gene on CFTR function. METHODS: Organoids of F508del/class I, F508del/S1251N and pwCF with only one detected CF-causing mutation were cultured. Allele-specific CFTR variation was investigated using targeted locus amplification (TLA), CFTR function was measured using the forskolin-induced swelling assay and mRNA levels were quantified using RT-qPCR. RESULTS: We were able to distinguish CFTR genotypes based on TLA data. Additionally, we observed heterogeneity within genotypes, which we were able to link to CFTR function for S1251N alleles. CONCLUSIONS: Our results indicate that the paired analysis of CFTR intragenic variation and CFTR function can gain insights in the underlying CFTR defect for individuals where the disease phenotype does not match the CFTR mutations detected during diagnosis.

T cells in cardiac allograft vasculopathy are skewed to memory Th-1 cells in the presence of a distinct Th-2 population.

Cardiac allograft vasculopathy (CAV) in heart transplantation (HTx) patients remains the major complication for long-term survival, due to concentric neointima hyperplasia induced by infiltrating mononuclear cells (MNC). Previously, we showed that activated memory T-helper-1 (Th-1) cells are the major component of infiltrating MNC in coronary arteries with CAV. In this study, a more detailed characterization of the MNC in human coronary arteries with CAV (n = 5) was performed and compared to coronary arteries without CAV (n = 5), by investigating MNC markers (CD1a, DRC-1, CD3, CD20, CD27, CD28, CD56, CD68, CD69, FOXP3 and HLA-DR), cytokines (IL-1A, 2, 4, 10, 12B, IFN-gamma, and TGF-beta1), and chemokine receptors (CCR3, CCR4, CCR5, CCR7, CCR8, CXCR3 and CX3CR1) by immunohistochemical double-labeling and quantitative PCR on mRNA isolated from laser microdissected layers of coronary arteries. T cells in the neointima and adventitia of CAV were skewed toward an activated memory Th-1 phenotype, but in the presence of a distinct Th-2 population. FOXP3 positive T cells were not detected and production of most cytokines was low or absent, except for IFN-gamma, and TGF-beta. This typical composition of T-helper cells and especially production of IFN-gamma and TGF-beta may play an important role in the proliferative CAV reaction.