Peter van Mourik

181 General discussion Codons (PTCs) can potentially be rescued using this approach, while no off-target effects were detected 8 . These results are very promising, since base editing is an efficient and precise approach and could work for mutations that are not amenable to currently available CFTR-modulator treatment. One of the major obstacles for gene therapy (i.e. inserting wild-type (WT) CFTR) is inefficiency of available vectors that transport the gene (or gene product) into the cell nucleus. Many trials have been conducted, and the only successful clinical trial could only detect a modest effect, most probably due to the use of a non-optimal vector and delivery method 9 . Intestinal organoids have been used to screen and validate different newly developed chimeric vectors 10 , and have been important in the selection and validation of an rAAV vector that is currently being evaluated in a clinical trial for patients with CF 11 . Another very promising strategy is the use of antisense oligonucleotides (AONs) to correct mRNA defects including alternative exon splicing sites 12,13 that are not amenable to available CFTR-modulators. AONs are clinically available for several diseases including Spinal Muscular Atrophy 14 , while an exciting new example of AON use has been in a patient suffering from Batten disease who had a unique mutation in the MFSD8 gene 15 . Using patient-derived cells an AON was specifically designed for this individual subject, and through expedited pre-clinical development, the patient received an effective drug within 1 year of discovering the genetic defect 15 . These results have sparked interest from many different research groups investigating a spectrum of diseases, and could be very interesting for further application in Cystic Fibrosis. As AONs need to be specifically designed for a mutation and validated pre- clinically before they can be introduced in the clinic, assessing whether the AONs improve CFTR function in organoids can be extremely helpful for rare mutations. The current HUB/UMCU biobank contains ~40 splice variants that could be targeted and could thus serve as a valuable resource for personalized AON development 8 , while other biobanks might also contain unique splice mutations. High-content drug screening to identify CFTR-modulating compounds The high proliferation rate of intestinal tissue not only allows for long-term culturing, but also creates the opportunity for large scale high-content screening. Previously, libraries of G-protein Coupled Receptor (GCPR) compounds were screened in organoids, which identified beta-adrenergic agonists as activators of CF both in vitro and in vivo 16 . Since these drugs are clinically available and are generally safe, a clinical trial testing both oral and inhaled formulations was conducted, finding limited usefulness mainly due to side effects of the drugs, although in vivo CFTR- 9