Fig. 2: ASO cocktail optimization and mechanism of action.
From: Gene-specific nonsense-mediated mRNA decay targeting for cystic fibrosis therapy
ASOs targeting CFTR (a) exon 24, (b) exon 25, or (c) exon 26 were individually screened in 16HBE-W1282X cells, in combination with two ASOs that target the other two exons, at a total nominal concentration of 120 nM. Red bars indicate the lead ASO identified in each combination screen. d Comparison between LC15-1 and LC15-2. CFTR and EIF4A2 mRNA levels in (e) 16HBE-G542X cells and (f) 16HBE-R1162X cells transfected with ASOs at a total nominal concentration of 120 nM. g The number of required EJCs targeted by ASOs C478, C495, C515, or all together, was assessed by transfecting 16HBE-W1282X cells with one, two, or three EJC-targeting ASOs at the same total nominal concentration. h CFTR mRNA in 16HBE-W1282X cells were transfected with various combinations of the lead ASOs C478, C495, and C515. i. Mean PSI of each CFTR isoform in 16HBE-W1282X cells transfected with various combinations of C478, C495, and C515. All mRNA levels in (a)–(g) were measured by RT-qPCR; CFTR mRNA levels were measured using forward and reverse primers targeting exon 22 and exon 23, respectively. RPL32 mRNA level served as an internal reference. PSI measured in (i) used forward and reverse primers targeting exon 23 and exon 27, respectively. Abbreviations are as in Fig. 1; LC15-2 = C478/494/514. Data are represented as mean values ± SD. All data points represent independent biological replicates. a, b, and d–f (n = 3). c (n = 6 except n = 5 for 478/494/526). h (n = 3 except n = 2 for LC15-1 [40:40:40]). g (n = 3 except n = 4 for NT, Sc15, CC15, and LC15-1 [40:40:40]). i (n = 4 except n = 3 for LC15-1 [120:0:0], [0:120:0], [0:0:120], [60:60:0], [0:60:60], and [60:0:60]). For all Dunnett’s post-tests, n.s. P > 0.05, */‡P < 0.05, **P < 0.01, ***P < 0.001. For all Student’s t-tests, #P < 0.05. a–h One-way ANOVA with Dunnett’s post-test versus NT, or Student’s t-test. h ([total ASO] = 70 nM): one-way ANOVA with Dunnett’s post-test vs LC15-1 [10:40:20]. i One-way ANOVA with Dunnett’s post-test, versus each isoform in ‘LC15-1 [40:40:40]’. Source data are provided as a Source Data File.
