Comprehensive Assessment of CFTR Modulators' Therapeutic Efficiency for N1303K Variant

Abstract

p.Asn1303Lys (N1303K) is a common missense variant of the CFTR gene, causing cystic fibrosis (CF). In this study, we initially evaluated the influence of CFTR modulators on the restoration of N1303K-CFTR function using intestinal organoids derived from four CF patients expressing the N1303K variant. The forskolin-induced swelling assay in organoids offered valuable insights about the beneficial effects of VX-770 + VX-661 + VX-445 (Elexacaftor + Tezacaftor + Ivacaftor, ETI) on N1303K-CFTR function restoration and about discouraging the prescription of VX-770 + VX-809 (Ivacaftor + Lumacaftor) or VX-770 + VX-661 (Ivacaftor + Tezacaftor) therapy for N1303K/class I patients. Then, a comprehensive assessment was conducted on an example of one patient with the N1303K/class I genotype to examine the ETI effect on the restoration of N1303K-CFTR function using in vitro the patient's intestinal organoids, ex vivo the intestinal current measurements (ICM) method and assessment of the clinical status before and after targeted therapy. All obtained results are consistent with each other and have proven the effectiveness of ETI for the N1303K variant. ETI produced a significant positive effect on forskolin-induced swelling in N1303K/class I organoids indicating functional improvement of the CFTR protein; ICM demonstrated that ETI therapy restored CFTR function in the intestinal epithelium after three months of treatment, and the patient improved his clinical status and lung function, increased his body mass index (BMI) and reduced the lung pathogenic flora diversity, surprisingly without improving the sweat test results.

Keywords: BMI; FEV1; N1303K; clinical status; cystic fibrosis; intestinal current measurements; intestinal organoids.

Figure 1

Morphology of intestinal organoids derived from CF patients with N1303Kvariant compared to c F508del/F508del and wt/wt CFTR cultures (controls). Five days in culture, 20× objective, scale bar: 200 μm.

Figure 2

Assessment of CFTR modulators’ effects on forskolin (Fsk) treatment in CF organoids with N1303K variant. Control–F508del/F508del organoids; each modulator concentration is 3.5 µM; ** p < 0.01, *** p < 0.001 compared to Fsk alone by unpaired Student’s t-test.

Figure 3

N1303K/2143delT, N1303K/3821delT, N1303K/G461E and F508del/F508del (control) intestinal organoid swelling upon 1 h treatment with 5 µM forskolin. Organoids were stained with Calcein AM, and each CFTR-modulator concentration is 3.5 µM; Scale bar: 500 μm.

Figure 4

Results of functional activity study of intestinal epithelial ion channels in N1303K/2143deT patient by ICM: (a) before the treatment with targeted therapy, there is no response to forskolin and a negative change of ΔI_SC upon carbachol and histamine application; (b) during the targeted therapy and 3 months later, a positive changed ΔI_SC was observed after forskolin, carbachol and histamine application. The plots in Figure 4a,b are representative, and each plot is the result of a short-circuit current measurement for one of the three studied biopsies.