Intestinal organoids as a platform for functional evaluation of ASO-mediated splicing modulation in cystic fibrosis

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.