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Updated: Jan 13, 2026
Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients
Published on: February 11, 2017
Intestinal organoids as a platform for functional evaluation of ASO-mediated splicing modulation in cystic fibrosis
Noemie Stanleigh1, Michal Irony-Tur Sinai1, Yifat Oren2
1Department of Genetics, the Hebrew University of Jerusalem, Israel.
Background:
CFTR modulating therapies have advanced significantly but remain ineffective for people with CF (pwCF) with mutations preventing CFTR protein production. PwCF carrying non-canonical splicing mutations show modest benefits. SPL84, an antisense oligonucleotide (ASO) developed by Splisense, targets the non-canonical CFTR splicing mutation 3849+10 kb C→T (3849) and is currently in Phase2 trials. SPL84-23, a longer SPL84 variant, was shown to modulate splicing and rescue CFTR activity in patient-derived respiratory epithelial cells carrying the 3849 mutation. Here, we investigated intestinal organoids as a platform to assess ASO-based splicing modulation.
Methods:
SPL84-23 effect on CFTR splicing was measured using RT-PCR and RT-qPCR. Functional rescue was assessed in 3D-organoids by forskolin-induced swelling, and in 2D-monolayers by short-circuit current assays.
Results:
We established a novel free-uptake ASO delivery protocol into 3D-intestinal organoids and 2D-monolayers from 3849 pwCF. All cultures showed high basal levels of correctly spliced transcripts, leading to high residual CFTR activity. SPL84-23 treatment reduced aberrant splicing by 78 %, in both 3D and 2D systems, resulting in significant improvements in CFTR function across all organoid cultures.
Conclusion:
Intestinal organoids, both 3D and monolayers, provide a suitable platform for assessing ASO-based splicing modulation. Our study further implies that the high level of correctly spliced 3849 CFTR transcripts in intestinal epithelial cells may contribute to the mild intestinal symptoms in pwCF carrying non-canonical splicing mutations.
Insights
Antisense oligonucleotide SPL84-23 effectively corrects CFTR splicing defects in intestinal organoids from cystic fibrosis patients with the 3849 mutation. This study validates intestinal organoids as a platform for evaluating CFTR splicing modulation therapies.
Area of Science:
- Biotechnology
- Genetics
- Molecular Biology
Background:
- Current cystic fibrosis (CF) therapies are ineffective for patients with mutations preventing CFTR protein production.
- Antisense oligonucleotides (ASOs) show promise for targeting specific CFTR mutations.
- SPL84-23, a variant of SPL84, has demonstrated potential in modulating CFTR splicing and rescuing function in patient-derived cells.
Purpose of the Study:
- To investigate intestinal organoids as a platform for assessing ASO-based CFTR splicing modulation.
- To evaluate the efficacy of SPL84-23 in correcting the 3849+10 kb C→T splicing mutation in CFTR.
Main Methods:
- Developed a novel free-uptake protocol for delivering ASOs into 3D-intestinal organoids and 2D-monolayers.
- Measured CFTR splicing modulation using RT-PCR and RT-qPCR.
- Assessed functional CFTR rescue via forskolin-induced swelling in organoids and short-circuit current assays in monolayers.
Main Results:
- Established a robust model using intestinal organoids from patients with the 3849 CFTR mutation.
- SPL84-23 treatment reduced aberrant CFTR splicing by 78% in both 3D and 2D systems.
- Demonstrated significant improvements in CFTR function across all treated organoid cultures.
Conclusions:
- Intestinal organoids (3D and monolayers) are suitable platforms for evaluating ASO-based splicing modulation therapies.
- The high residual correctly spliced CFTR transcripts in intestinal cells may explain milder intestinal symptoms in patients with the 3849 mutation.
