CFTR modulator therapy for cystic fibrosis caused by the rare c.3700A>G mutation.

BACKGROUND: The c.3700A>G mutation, a rare cystic fibrosis (CF)-causing CFTR mutation found mainly in the Middle East, produces full-length transcript encoding a missense mutation (I1234V-CFTR), and a cryptic splice site that deletes 6 amino acids in nucleotide binding domain 2 (I1234del-CFTR). METHODS: FRT cell models expressing I1234V-CFTR and I1234del-CFTR were generated. We also studied an I1234del-CFTR-expressing gene-edited human bronchial (16HBE14o-) cell model, and primary cultures of nasal epithelial cells from a c.3700A>G homozygous subject. To identify improved mutation-specific CFTR modulators, high-throughput screening was done using I1234del-CFTR-expressing FRT cells. Motivated by the in vitro findings, Trikafta was tested in two c.3700A>G homozygous CF subjects. RESULTS: FRT cells expressing full-length I1234V-CFTR had similar function to that of wildtype CFTR. I1234del-CFTR showed reduced activity, with modest activation seen with potentiators VX-770 and GLPG1837, correctors VX-809, VX-661 and VX-445, and low-temperature incubation. Screening identified novel arylsulfonyl-piperazine and spiropiperidine-quinazolinone correctors, which when used in combination with VX-445 increased current ~2-fold compared with the VX-661/VX-445 combination. The combination of VX-770 with arylsulfonamide-pyrrolopyridine, piperidine-pyridoindole or pyrazolo-quinoline potentiators gave 2-4-fold greater current than VX-770 alone. Combination potentiator (co-potentiator) efficacy was also seen in gene-edited I1234del-CFTR-expressing human bronchial epithelial cells. In two CF subjects homozygous for the c.3700A>G mutation, one subject had a 27 mmol/L decrease in sweat chloride and symptomatic improvement on Trikafta, and a second subject showed a small improvement in lung function. CONCLUSIONS: These results support the potential benefit of CFTR modulators, including co-potentiators, for CF caused by the c.3700A>G mutation.

1-BENZYLSPIRO[PIPERIDINE-4,1'-PYRIDO[3,4-b]indole] 'co-potentiators' for minimal function CFTR mutants.

We previously identified a spiro [piperidine-4,1-pyrido [3,4-b]indole] class of co-potentiators that function in synergy with existing CFTR potentiators such as VX-770 or GLGP1837 to restore channel activity of a defined subset of minimal function cystic fibrosis transmembrane conductance regulator (CFTR) mutants. Here, structure-activity studies were conducted to improve their potency over the previously identified compound, 20 (originally termed CP-A01). Targeted synthesis of 37 spiro [piperidine-4,1-pyrido [3,4-b]indoles] was generally accomplished using versatile two or three step reaction protocols with each step having high efficiency. Structure-activity relationship studies established that analog 2i, with 6'-methoxyindole and 2,4,5-trifluorobenzyl substituents, had the greatest potency for activation of N1303K-CFTR, with EC50 ∼600 nM representing an ∼17-fold improvement over the original compound identified in a small molecule screen.

Preclinical Studies of a Rare CF-Causing Mutation in the Second Nucleotide Binding Domain (c.3700A>G) Show Robust Functional Rescue in Primary Nasal Cultures by Novel CFTR Modulators.

The combination therapies ORKAMBITM and TRIKAFTATM are approved for people who have the F508del mutation on at least one allele. In this study we examine the effects of potentiator and corrector combinations on the rare mutation c.3700A>G. This mutation produces a cryptic splice site that deletes six amino acids in NBD2 (I1234-R1239del). Like F508del it causes protein misprocessing and reduced chloride channel function. We show that a novel cystic fibrosis transmembrane conductance regulator CFTR modulator triple combination (AC1, corrector, AC2-2, co-potentiator and AP2, potentiator), rescued I1234-R1239del-CFTR activity to WT-CFTR level in HEK293 cells. Moreover, we show that although the response to ORKAMBI was modest in nasal epithelial cells from two individuals homozygous for I1234-R1239del-CFTR, a substantial functional rescue was achieved with the novel triple combination. Interestingly, while both the novel CFTR triple combination and TRIKAFTATM treatment showed functional rescue in gene-edited I1234-R1239del-CFTR-expressing HBE cells and in nasal cells from two CF patients heterozygous for I1234-R1239del/W1282X, nasal cells homozygous for I1234-R1239del-CFTR showed no significant response to the TRIKAFTATM combination. These data suggest a potential benefit of CFTR modulators on the functional rescue of I1234-R1239del -CFTR, which arises from the rare CF-causing mutation c.3700A>G, and highlight that patient tissues are crucial to our full understanding of functional rescue in rare CFTR mutations.

Orkambi® and amplifier co-therapy improves function from a rare CFTR mutation in gene-edited cells and patient tissue.

The combination therapy of lumacaftor and ivacaftor (Orkambi®) is approved for patients bearing the major cystic fibrosis (CF) mutation: ΔF508 It has been predicted that Orkambi® could treat patients with rarer mutations of similar "theratype"; however, a standardized approach confirming efficacy in these cohorts has not been reported. Here, we demonstrate that patients bearing the rare mutation: c.3700 A>G, causing protein misprocessing and altered channel function-similar to ΔF508-CFTR, are unlikely to yield a robust Orkambi® response. While in silico and biochemical studies confirmed that this mutation could be corrected and potentiated by lumacaftor and ivacaftor, respectively, this combination led to a minor in vitro response in patient-derived tissue. A CRISPR/Cas9-edited bronchial epithelial cell line bearing this mutation enabled studies showing that an "amplifier" compound, effective in increasing the levels of immature CFTR protein, augmented the Orkambi® response. Importantly, this "amplifier" effect was recapitulated in patient-derived nasal cultures-providing the first evidence for its efficacy in augmenting Orkambi® in tissues harboring a rare CF-causing mutation. We propose that this multi-disciplinary approach, including creation of CRISPR/Cas9-edited cells to profile modulators together with validation using primary tissue, will facilitate therapy development for patients with rare CF mutations.