Evaluating “exon‐skipping” as a strategy to repair the CFTR nonsense mutation

PI: Christine Bear, PhD

Institution: Sickkids Hospital - Toronto

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Background, Rationale and Research Goals: Currently, there is a paucity of relevant cell- based assays for studying the efficacy of novel investigational compounds targeting rare CF causing mutations. In our view, the molecular defects caused by c.3846G>A (W1282X) are complex wherein the molecular phenotypes, ie. reduced stability of the aberrant transcript and the reduced functional expression of the aberrant protein cannot be recapitulated with fidelity in heterologous expression systems. Hence, modulators of W1282X should be testing in patient- derived tissue.  Furthermore- this testing platform should be scalable to a 96 well plate format so that a panel of modulators can be tested simultaneously. We developed and published a fluorescence based assay for the study of CFTR modulators in patient-derived nasal epithelial cells and showed in small scale- proof of concept studies- that this method could also be applied to the study of patient-derived iPSCs differentiated to lung using protocol by Wong et al. (https://www.ncbi.nlm.nih.gov/pubmed/22922672). Our first goal, with partial funding from the Bike Ride Grant, was to determine the feasibility of increasing the scale of this fluorescence based assay of CFTR channel activity to lung cells differentiated from non-CF stem cells. In the longer term- we plan to validate this assay to study modulator activity in lung cells differentiated from iPSCs from individuals bearing the c.3846G>A (W1282X) mutation.

Research Results: As previously mentioned, we developed a FLiPR- plate reader assay to monitor CFTR mediated depolarization in nasal epithelial cultures. (https://www.ncbi.nlm.nih.gov/pubmed/28649446) The funding provided by the Bike Ride Award contributed to our development and optimization of a 96 well, FLiPR assay of CFTR channel activity in CA1-embrionic stem cells differentiated to immature lung cells (figure below shows goodness of the assay). This robust assay will now be tested in studies of emerging modulators of W1282X using IPSCs from patients with this mutation and generated at SickKids in Toronto through a Program co-sponsored by CF Canada

(link: http://www.sickkids.ca/cysticfibrosiscentre/Individualized-Therapy/Index.html)

bear graph page 2

This graph shows high Z factor for high‐ throughput (96 well) fluorescence assay of CFTR channel function (measured using FLiPR) in Stage 3 and 4 lung differentiated from non‐CF ES cells. These data support our claim that a panel of investigational compounds can be profiled for efficacy in modulating CFTR function using this assay. Hits on the assay using progenitor/ immature lung cells will be validated on fully differentiated lung epithelial cultures.