Functional Restoration of CFTR Nonsense Mutations in Intestinal Organoids

Abstract

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-02disulfate 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.

One Size Does Not Fit All: The Past, Present and Future of Cystic Fibrosis Causal Therapies

“…Several strategies have been proposed in pre-clinical models to prevent NMD and enhance mRNA expression of PTC CFTR (reviewed in [ 266 , 267 ]). Inhibition of the NMD activator serine/threonine-protein kinase SMG1, via the small molecule inhibitor SMG1i, resulted in increased levels of G542X, W1282X and other PTC containing mRNAs in various models, including airway epithelial cells, primary rectal organoids and several CF animal models [ 58 , 268 , 269 , 270 , 271 , 272 , 273 , 274 ]. As SMG1 is active in other cellular processes besides NMD, its inhibition causes considerable toxicity, which likely precludes it from translation into a therapy [ 272 , 275 ].…”

“…Similarly, G542X appears to be more responsive to TRIDS than W1282X due to differences in the respective sequence contexts [ 311 ]. To rescue the resulting missense mutations, many pre-clinical studies now focus on combining TRIDs with other classes of compounds, which in most cases synergistically improves functional rescue [ 58 , 263 , 268 , 269 , 270 , 271 , 272 , 273 , 274 , 277 ].…”

High-Throughput Functional Assay in Cystic Fibrosis Patient-Derived Organoids Allows Drug Repurposing

“…Z'-factors were robust in the W1282X/W1282X screen as well, however, the positive control here consisted of F508del/S1251N organoids treated with VX-770 as a more suitable positive control specifically for W1282X/W1282X organoids was not available. Potentially in future studies the recently described RT agent DAP can be used for this 22 or a combination of compounds that together results in higher levels of CFTR rescue 8 . In the future, additional automation such as automated organoid dispensers, drug printers and centrifugal washers might further reduce technical variability, thereby increasing the Z'-factor.…”

“…A novel recently developed RT agent ELX-02 (NB124; Eloxx Pharmaceuticals) resulted in production of full length CFTR protein and restoration of CFTR function in G542X/G542X intestinal organoids 7 . However, in a study characterizing the effect of ELX-02 in a larger set of patient-derived organoids, ELX-02 as single agent resulted in only limited restoration of CFTR function 8 . In a recent press release by Eloxx Pharmaceuticals, first results of their phase 2 trial in homozygous G542X CF patients describe only a minor decrease in sweat chloride levels ('Eloxx Pharmaceuticals Reports', 2021).…”

Bicarbonate Transport in Cystic Fibrosis and Pancreatitis

“…Modulators are pharmacological compounds that can be classified on the basis of their different modes of action. So-called potentiators, e.g., ivacaftor (VX-770), restore CFTR channel gating; correctors, e.g., lumacaftor (VX-809), tezacaftor (VX-661) and elexacaftor (VX-445), improve CFTR folding and trafficking to the cell surface, and some such as elexacaftor may also act as co-potentiators [ 133 ]; amplifiers, e.g., PTI-CH, co-translationally enhance CFTR biosynthesis by stabilizing CFTR mRNA [ 134 ]; and read-through agents for CFTR nonsense mutations, e.g., aminoglycosides [ 135 ] and analogs (ELX-02) [ 136 , 137 ], or the recently developed compound SRI-37240, suppress premature termination codons [ 138 ]. The spectrum of mutations for which each modulator is clinically approved was reviewed recently [ 139 ].…”