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Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients
Published on: February 11, 2017
Functional Restoration of CFTR Nonsense Mutations in Intestinal Organoids
E de Poel1, S Spelier2, S W F Suen2
1Department of Pediatric Respiratory Medicine, Wilhelmina Children's Hospital, University Medical Center, Utrecht University, 3584 EA Utrecht, The Netherlands; Regenerative Medicine Utrecht, University Medical Center, Utrecht University, 3584 CT Utrecht, The Netherlands; Center for Living Technologies, Eindhoven-Wageningen-Utrecht Alliance, The Netherlands.
Background:
Pharmacotherapies for people with cystic fibrosis (pwCF) who have premature termination codons (PTCs) in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are under development. Thus far, clinical studies focused on compounds that induce translational readthrough (RT) at the mRNA PTC location. Recent studies using primary airway cells showed that PTC functional restoration can be achieved through combining compounds with multiple mode-of-actions. Here, we assessed induction of CFTR function in PTC-containing intestinal organoids using compounds targeting RT, nonsense mRNA mediated decay (NMD) and CFTR protein modulation.
Methods:
Rescue of PTC CFTR protein was assessed by forskolin-induced swelling of 12 intestinal organoid cultures carrying distinct PTC mutations. Effects of compounds on mRNA CFTR level was assessed by RT-qPCRs.
Results:
Whilst response varied between donors, significant rescue of CFTR function was achieved for most donors with the quintuple combination of a commercially available pharmacological equivalent of the RT compound (ELX-02-disulfate or ELX-02ds), NMD inhibitor SMG1i, correctors VX-445 and VX-661 and potentiator VX-770. The quintuple combination of pharmacotherapies reached swelling quantities higher than the mean swelling of three VX-809/VX-770-rescued F508del/F508del organoid cultures, indicating level of rescue is of clinical relevance as VX-770/VX-809-mediated F508del/F508del rescue in organoids correlate with substantial improvement of clinical outcome.
Conclusions:
Whilst variation in efficacy was observed between genotypes as well as within genotypes, the data suggests that strong pharmacological rescue of PTC requires a combination of drugs that target RT, NMD and protein function.
Insights
Combining drugs targeting multiple mechanisms significantly restored cystic fibrosis transmembrane conductance regulator (CFTR) function in organoids with premature termination codons (PTCs). This approach shows promise for treating cystic fibrosis (CF) patients with PTC mutations.
Area of Science:
- Genetics and Molecular Biology
- Pharmacology
- Cell Biology
Background:
- Development of pharmacotherapies for cystic fibrosis (CF) patients with premature termination codons (PTCs) in the CFTR gene is ongoing.
- Current clinical studies focus on translational readthrough (RT) compounds.
- Combining multiple modes of action, including RT, nonsense mRNA mediated decay (NMD) inhibition, and CFTR protein modulation, can restore PTC CFTR function.
Purpose of the Study:
- To assess the induction of CFTR function in intestinal organoids with PTC mutations.
- To evaluate the efficacy of drug combinations targeting RT, NMD, and CFTR protein modulation.
Main Methods:
- Utilized forskolin-induced swelling assays in 12 intestinal organoid cultures with distinct PTC mutations to assess CFTR protein rescue.
- Quantified CFTR mRNA levels using RT-qPCR to evaluate compound effects.
Main Results:
- A quintuple combination therapy (ELX-02ds, SMG1i, VX-445, VX-661, VX-770) significantly rescued CFTR function across most donors.
- The combination therapy achieved higher swelling quantities than established F508del/F508del organoid rescue models, indicating clinical relevance.
- Observed variations in efficacy based on genotype and donor, but overall significant rescue was achieved.
Conclusions:
- Effective pharmacological rescue of PTC CFTR function necessitates a combination of drugs targeting RT, NMD, and protein function.
- This multi-targeted approach demonstrates potential for treating CF patients with PTC mutations.
- Further research is warranted to optimize combination therapies for diverse CFTR PTC genotypes.
