Protocol for Application, Standardization and Validation of the Forskolin-Induced Swelling Assay in Cystic Fibrosis Human Colon Organoids

Vonk, Annelotte M.; Mourik, Peter van; Ramalho, A.S.; Silva, Iris; Statia, Marvin; Kruisselbrink, Evelien; Suen, S.W.F.; Dekkers, Johanna F.; Vleggaar, Frank P.; Houwen, Roderick H. J.; Mullenders, Jasper; Boj, Sylvia F.; Vries, Robert R G; Amaral, Margarida D.; Boeck, K. De; Ent, Cornelis K. van der; Beekman, Jeffrey M.

doi:10.1016/j.xpro.2020.100019

Cited by 86 publications

(103 citation statements)

References 6 publications

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“…Human rectal biopsies were obtained by rectoscopy and forceps biopsy as described previously [ 30 , 35 , 36 ]. The tissue was mounted on a circulating micro-Ussing chamber as described previously [ 32 , 33 ].…”

Section: Methodsmentioning

confidence: 99%

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Personalized Medicine Based on Nasal Epithelial Cells: Comparative Studies with Rectal Biopsies and Intestinal Organoids

Silva

Railean

Duarte

et al. 2021

JPM

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As highly effective CFTR modulator therapies (HEMT) emerge, there is an unmet need to find effective drugs for people with CF (PwCF) with ultra-rare mutations who are too few for classical clinical trials and for whom there are no drug discovery programs. Therefore, biomarkers reliably predicting the benefit from CFTR modulator therapies are essential to find effective drugs for PwCF through personalized approaches termed theranostics. Here, we assess CFTR basal function and the individual responses to CFTR modulators in primary human nasal epithelial (pHNE) cells from PwCF carrying rare mutations and compare these measurements with those in native rectal biopsies and intestinal organoids, respectively, in the same individual. The basal function in pHNEs shows good correlation with CFTR basal function in rectal biopsies. In parallel, CFTR rescue in pHNEs by CFTR modulators correlates to that in intestinal organoids. Altogether, results show that pHNEs are a bona fide theranostic model to assess CFTR rescue by CFTR modulator drugs, in particular for PwCF and rare mutations.

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Section: Methodsmentioning

confidence: 99%

“…Intestinal organoids were obtained by crypt isolation from rectal biopsies and seeded in 50% Matrigel as described by Vonk et al [ 36 ]. The organoids were passaged ~1 per week, and the medium was refreshed every 2–3 days.…”

Section: Methodsmentioning

confidence: 99%

Personalized Medicine Based on Nasal Epithelial Cells: Comparative Studies with Rectal Biopsies and Intestinal Organoids

Silva

Railean

Duarte

et al. 2021

JPM

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“…Collection, isolation, expansion biobanking procedures, and FIS evaluation of PDOs were conducted as recently described [12] with modifications to accommodate the mechanism of action being ) Following induction with forskolin, PDOs treated with either vehicle or ELX-02 were imaged every 10 min for 120 min; representative images demonstrating change in size relative to t = 0 min and t = 120 min are presented. ( B. )…”

Section: Pdo Fis Assaymentioning

confidence: 99%

Targeting G542X CFTR nonsense alleles with ELX-02 restores CFTR function in human-derived intestinal organoids

Crawford

Mullenders²,

Pott³

et al. 2021

Journal of Cystic Fibrosis

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Background: Promoting full-length protein production is a requisite step to address some of the remaining unmet medical need for those with Cystic Fibrosis (CF) nonsense alleles. ELX-02 promotes readthrough of mRNA transcripts bearing nonsense mutations, including the most common CF nonsense allele G542X , in several different preclinical models including human bronchial epithelial cells. Here we evaluate ELX-02 mediated read-through using the CFTR-dependent Forskolin-induced swelling (FIS) assay across a selection of G542X genotype patient derived organoids (PDOs). Methods: CFTR functional restoration was evaluated in ELX-02 treated G542X homozygous and heterozygous PDOs in the CFTR-dependent FIS assay. CFTR mRNA abundance and integrity were evaluated by qPCR and Nanostring analysis while PDO protein was detected by capillary based size-exclusion chromatography. Results: PDOs homozygous for G542X or heterozygous with a second minimally functional allele had significantly increased CFTR activity with ELX-02 in a dose-dependent fashion across a variety of forskolin induction concentrations. The functional increases are similar to those obtained with tezacaftor/ivacaftor in F508del homozygous PDOs. Increased CFTR C-and B-band protein was observed in accordance with increased function. In addition, ELX-02 treatment of a G542X/G542X PDO results in a 5-fold increase in CFTR mRNA compared with vehicle treated, resulting in normalization of CFTR mRNA as measured via Nanostring. Conclusions: These data with ELX-02 in PDOs are consistent with previous G542X model evaluations. These results also support the ongoing clinical evaluation of ELX-02 as a read-through agent for CF caused by the G542X allele.

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“…The generation of intestinal organoids from biopsies and subsequent fluid secretion assays (FIS-assays) were performed according to a previously described protocol 34 . All FIS-assay experiments were conducted in duplicate and for the majority of the donors at multiple culturing time points with a maximum of 7 consecutive culture time points (n=7).…”

Section: Methodsmentioning

confidence: 99%

Forskolin-induced swelling of intestinal organoids predicts long-term cystic fibrosis disease progression

Muilwijk

Mourik

et al. 2021

Preprint

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Patient-derived organoids hold great potential as predictive biomarker for disease expression or therapeutic response. Here, we used intestinal organoids to estimate individual cystic fibrosis transmembrane conductance regulator (CFTR) function of people with cystic fibrosis, a monogenic life-shortening disease associated with more than 2000 CFTR mutations and highly variable disease progression. In vitro CFTR function in CF intestinal organoids of 176 individuals with diverse CFTR mutations was quantified by forskolin induced swelling and was strongly associated with longitudinal changes of lung function and development of pancreatic insufficiency, CF-related liver disease and diabetes. This association was not observed when the commonly used biomarker of CFTR function sweat chloride concentration was used. The data strongly exemplifies the value of an organoid-based biomarker in a clinical disease setting and supports the prognostic value of forskolin induced swelling of intestinal organoids, especially for people with CF who have rare CFTR genotypes with unclear clinical consequences.

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Protocol for Application, Standardization and Validation of the Forskolin-Induced Swelling Assay in Cystic Fibrosis Human Colon Organoids

Cited by 86 publications

References 6 publications

Personalized Medicine Based on Nasal Epithelial Cells: Comparative Studies with Rectal Biopsies and Intestinal Organoids

Personalized Medicine Based on Nasal Epithelial Cells: Comparative Studies with Rectal Biopsies and Intestinal Organoids

Targeting G542X CFTR nonsense alleles with ELX-02 restores CFTR function in human-derived intestinal organoids

Forskolin-induced swelling of intestinal organoids predicts long-term cystic fibrosis disease progression

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