Background/ Concept

Cystic fibrosis (CF) is the most common of all rare, lethal autosomal-recessive diseases with an average incidence in the EU of one in 2000-3000 newborns affected. CF is caused by mutations in the CFTR gene, a cAMP-regulated apical chloride channel of secretory epithelia in the airways, pancreas, intestine, bile duct and other tissues. Nearly 2000 CFTR gene mutations are known, of which ~130 are pathogenic, impairing its translation, cellular processing, and/or chloride channel gating. Therefore, small molecule therapy restoring function to mutant CFTR is a priority in the field. High throughput (HT) screens have identified CFTR potentiators, which restore the channel activity by enhancing gating, and correctors, which rescue the most frequent trafficking mutant (F508del) to the cell surface in vitro. The potentiator Ivacaftor (VX-770) was approved by FDA / EMEA in 2013 for G551D and, recently for further eight gating mutations. However, these mutations are present in just 4-5% of all CF patients.

For most CF patients, an effective small molecule treatment is not yet available. Thus far, results from clinical trials on patients homozygous for F508del with the best known corrector drugs (VX-809/661) are modest. A combination of the correctors VX-809/661 and the potentiator VX-770 did improve lung function but only to a limited extent (~4%), and reportedly only in a subgroup of patients. Moreover, only ~40% of patients are F508del-homozygous and the efficacy of correctors for patients with only one F508del allele is expected to be lower. At least 15% of all CF patients are unlikely to benefit from F508del-CFTR corrector therapies, as they lack F508del in both alleles. As a result, there is clearly an unmet need for novel correctors of F508del-CFTR, and in particular for other rare trafficking (class II) mutants. Also, it has to be emphasized that current large screening initiatives based on primary airway cells are not able to target rare CF mutations, since explant lung tissue homozygous for rare mutations is generally not available. Only a single patient may be available for INSTINCT that is homozygous for the selected trafficking mutation A561E, which is the second most frequent mutation in Portugal (6.37%). This would allow us to collect cells for a secondary screening but it is not sufficient for a primary screen. A561E can be partially rescued by VX-809 in primary cultures of CF lung cells. However, in view of the limited clinical efficacy of VX-809 in homozygous F508del CFTR patients, we do not expect it to show higher efficacy in heterozygous A561E patients. Therefore additional screening is mandatory for these patients. In contrast, as typical for rare mutations, no patient is available homozygous for N1303K, which is another CF mutation with a severe trafficking defect, although it is relatively common in Southern Europe with allele frequencies up to 5%. Importantly, the N1303K defect is insensitive to F508del CFTR correctors like VX809 and corr-4a, as the protein alteration differs from that of F508del. Therefore, N1303K-CFTR will probably require treatment with different types of correctors or corrector combinations. Thus, it is clear that in the case of F508del and other trafficking mutations novel compounds have to be identified. Indeed, current data indicate that a combination of CFTR correctors, potentiators, and molecules that prevent an excessive turnover of mutant proteins will be required. This combination ideally should be tailored not only to specific CFTR mutations, but also to the individual patient, who in most cases presents two different mutations. Furthermore, the complexity of the mutant CFTR maturation and turnover kinetics requires the use of advanced cellular models that closely recapitulate the properties of the most affected organs (lung, bile ducts, pancreas and intestine). Ideally, this should be implemented at the screening stage to rapidly filter out compounds that are ineffective or toxic to the human native epithelium. However, primary culture techniques are cumbersome and, despite recent progress, airway cells of rare mutants, which are usually available only through bronchial brushes, will probably not provide enough material for high-throughput (HT) assays. More importantly, the genetic engineering of differentiated primary cells to establish appropriate reporter lines is extremely difficult.

Most screens have therefore been performed using immortalized cell lines overexpressing CFTR mutants and halide indicators. Only the most promising compounds were validated on primary human bronchial epithelial cells and showed highly variable and limited correction. However, the validation of compounds in stable organotypic cell systems at an early stage is essential, as immortalized cell lines do not show the physiological characteristics of the relevant respiratory, intestinal, pancreatic or bile duct epithelia, including regulation of CFTR expression, traffic and function, in particular in the context of inflammation and tissue injury responses. It is therefore not surprising that immortalized cell lines overexpressing mutant CFTR variants are poor predictors of clinical efficacy, and that alternative screening methods are required.


With the major breakthrough presented by induced pluripotent stem cells (iPSCs), another patient and disease-specific cell source for HT screens is now available. In contrast to primary bronchial cells, which presently provide the standard in differentiated airway cell culture, iPSCs show an unlimited potential for proliferation and differentiation. iPSCs can be generated from easily accessible cell sources, and major advances in mass production and targeted differentiation of such cells have been achieved. Of relevance for the project INSTINCT is the possibility to apply novel site-specific gene editing in hiPSCs including TALE Nucleases (TALENs). This opens up the unique possibility to generate patient and CFTR mutation-specific cell lineages, carrying a halide sensitive Yellow Fluorescent Protein (YFP), or a CFTR trafficking reporter system. Importantly, and in contrast to current screening initiatives based on primary cells or intestinal organoids, our approach of inactivating one (F508del) allele in compound heterozygous CF iPSCs through insertion of a CFTR reporter while leaving the allele of interest with the rare mutation untouched, generally offers the unique possibility for specifically screen for correctors of rare trafficking mutations, and to validate promising compounds in iPSC derived organotypic epithelia. Moreover, while gene editing in intestinal organoids currently requires antibiotic selection to obtain transgenic stem cell clones, we demonstrated footprintless gene editing of isogenic iPSC lines through TALEN / single stranded Oligonucleotide (ssODN) without antibiotic selection or FACSorting. This offers not only the opportunity to generate isogenic control lines by correcting or introducing mutations into the CFTR gene, but also to edit genomic modifiers of CF disease.

Our unique reporter iPSCs can be differentiated into CF relevant cell types such as respiratory, pancreatic and bile duct epithelium and utilized for HT screens of small molecules. Also, these iPSC-derivatives can be used for the further validation of compound candidates or combinations of compounds in novel electrophysiological and organotypic cell culture assays that probe more distant parameters, such as mucus secretion, bioactive lipid metabolism, inflammation and tissue remodeling. Multiwell automated confocal screening platforms are available for this purpose. On the long term, respiratory derivatives of gene-corrected patient-specific iPSCs may enable the realization of new concepts of personalized ex vivo gene therapy of CF.

We thus propose to use genetically engineered patient-specific iPS cells together with CF-specific primary lung epithelia as a valuable new platform for a better understanding of the different CF disease phenotypes, and for the identification of drugs that functionally correct the organ-specific phenotype of different (especially rare) CFTR trafficking mutations.