Peter van Mourik

172 Chapter 8 and preferably batches of reagents will be obtained by all labs to ensure the most optimal standardisation. Drugs to be tested The screening of the organoids will be divided up based on which drugs are being tested. Flatley Discovery Labs (FDL) is developing CFTR correctors and potentiators, and combinations of these drugs will be tested in the screen. Proteostasis Therapeutics (PTI) is developing CFTR correctors, potentiators and an amplifier, which will all be tested in the drug screen. Drugs from FDL and PTI will be tested within the same screen because of their comparable modes-of- action. Eloxx Pharmaceuticals is developing a CFTR read-through drug that will be screened separately, since the distinct mode-of-action necessitates different assay conditions. Moreover, the drugs developed by FDL and PTI are not expected to work on premature termination codons. Therefore, only organoids with maximum one PTC will be screened for responsiveness to FDL and PTI drugs, and organoids with at least one PTC will be screened for efficacy of ELOXX drugs. In both screens, organoids propagated in 24-wells plates will be transferred to 96- well plates for drug testing. Each experimental 96-wells plate will first go through several quality control checks to ensure that no pipetting errors have been made and the organoid cultures are of good quality by using reference organoid lines. These reference organoid lines are well characterized and the response of these reference organoids to the drugs from FDL, PTI and Eloxx should be within the range of what we have detected previously. CFTR function and response to the different drugs is measured by time-lapse confocal microscopy as the relative increase in surface area (as area under the curve, AUC) of calcein-green labelled organoids at 10 minute intervals for 60 minutes after stimulation with forskolin. Technological duplicates are measured for each condition, and individual drug responses are corrected for residual CFTR function upon forskolin stimulation. For FDL and PTI compounds, F508del homozygous organoids will be included in each experiment for comparison. For ELOXX compounds, G542X homozygous organoids will be included in each experiment for comparison. The best responding 27 organoids per lead product will be eligible for the clinical trials. SNP fingerprinting analysis and CFTR mutation sequencing As part of HUB’s quality control procedures, to confirm that no swap of material occurred during the whole screening process, genetic material from the biopsy tissue and the generated organoids will be isolated and submitted to SNP fingerprinting analysis. The obtained genetic material will be also used to confirm the CFTR