The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene variant, c.3453G > C (D1152H), is associated with mild Cystic Fibrosis (CF) disease, though there is considerable clinical variability ranging from no detectable symptoms to lung disease with early acquisition of Pseudomonas aeruginosa. The approval extension of ivacaftor, the first CFTR modulator drug approved, to include D1152H was based on a positive drug response of defective CFTR-D1152H chloride channel function when expressed in FRT cells. Functional analyses of primary human nasal epithelial cells (HNE) from an individual homozygous for D1152H now revealed that while CFTR-D1152H demonstrated normal, wild-type level chloride conductance, its bicarbonate-selective conductance was impaired. Treatment with ivacaftor increased this bicarbonate-selective conductance. Extensive genetic, protein and functional analysis of the nasal cells of this D1152H/D1152H patient revealed a 90% reduction of CFTR transcripts due to the homozygous presence of the 5T polymorphism in the poly-T tract forming a complex allele with D1152H. Thus, we confirm previous observation in patient-derived tissue that 10% normal CFTR transcripts confer normal, wild-type level chloride channel activity. Together, this study highlights the benefit of patient-derived tissues to study the functional expression and pharmacological modulation of CF-causing mutations, in order to understand pathogenesis and therapeutic responses.

The combination therapies ORKAMBI^TM and TRIKAFTA^TM 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 TRIKAFTA^TM 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 TRIKAFTA^TM 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.

Highly effective drugs modulating the defective protein encoded by the CFTR gene have revolutionized cystic fibrosis (CF) therapy. Preclinical drug-testing on human nasal epithelial (HNE) cell cultures and 3-dimensional human intestinal organoids (3D HIO) are used to address patient-specific variation in drug response and to optimize individual treatment for people with CF. This study is the first to report comparable CFTR functional responses to CFTR modulator treatment among patients with different classes of CFTR gene variants using the three methods of 2D HIO, 3D HIO, and HNE. Furthermore, 2D HIO showed good correlation to clinical outcome markers. A larger measurable CFTR functional range and access to the apical membrane were identified as advantages of 2D HIO over HNE and 3D HIO, respectively. Our study thus expands the utility of 2D intestinal monolayers as a preclinical drug testing tool for CF.

It has been suggested that in vitro studies of the rescue effect of CFTR modulator drugs in nasal epithelial cultures derived from people with cystic fibrosis have the potential to predict clinical responses to the same drugs. Hence, there is an interest in evaluating different methods for measuring in vitro modulator responses in patient-derived nasal cultures. Commonly, the functional response to CFTR modulator combinations in these cultures is assessed by bioelectric measurements, using the Ussing chamber. While this method is highly informative, it is time-consuming. A fluorescence-based, multi-transwell method for assaying regulated apical chloride conductance (Fl-ACC) promises to provide a complementary approach to theratyping in patient-derived nasal cultures. In the present work, we compared Ussing chamber measurements and fluorescence-based measurements of CFTR-mediated apical conductance in matching, fully differentiated nasal cultures derived from CF patients, homozygous for F508del (n = 31) or W1282X (n = 3), or heterozygous for Class III mutations G551D or G178R (n = 5). These cultures were obtained through a bioresource called the Cystic Fibrosis Canada-Sick Kids Program in Individual CF Therapy (CFIT). We found that the Fl-ACC method was effective in detecting positive responses to interventions for all genotypes. There was a correlation between patient-specific drug responses measured in cultures harbouring F508del, as measured using the Ussing chamber technique and the fluorescence-based assay (Fl-ACC). Finally, the fluorescence-based assay has the potential for greater sensitivity for detecting responses to pharmacological rescue strategies targeting W1282X.

Previous studies reported the influence of cis variants in F508del cystic fibrosis (CF) patients in their responses to CFTR modulators. The current study is a prospective, observational study involving three patients with CF and pancreatic insufficiency, carrying a complex allele including F508del with A238V, I1027T, or L467F. We report clinical data before and after 4 weeks of treatment with tezacaftor (TEZ)/ivacaftor (IVA), elexacaftor (ELX)/TEZ/IVA, and lumacaftor (LUM)/IVA for patients with complex alleles A238V, I1027T, and L467F, respectively. The 50-year-old patient bearing F508del;A238V/D1152H showed a normal sweat test (13 mEq/L) and improvements in forced expiratory volume in the first second (FEV₁) (+7 points), body mass index (BMI) (+0.85), and respiratory CF Questionnaire-Revised (CFQ-R) domain (+22.2 points). The 12-year-old patient bearing F508del;I1027T/R709X showed an improvement in a sweat test (−40 mEq/l), FEV₁ (+9 points) and the respiratory CFQ-R domain (+16.7 points). No changes in outcomes were observed for the 6-year-old patient F508del;L467F/F508del. Our data highlight that the reported variants do not modify the phenotypic expression of F508del. Searching L467F is crucial in CF patients with F508del nonresponsive to ELX/TEZ/IVA. Further data are needed to evaluate the clinical effect of these variants after a longer follow up.