*2.6. VX-809 Jointly with RE and RI Removal Completely Restored F508del-CFTR Function as Chloride Channel*

To investigate the channel function of the ∆RES- and ∆RIL-F508del-CFTR variants, we used the iodide efflux technique. Removal of RE<sup>S</sup> alone had no impact on F508del-CFTR function (Figure 5A; black dash line in Figure S3B) as expected from the lack of processed form for this variant (Figure 1B). However, when RE<sup>S</sup> and RI<sup>L</sup> were removed jointly from F508del-CFTR, functional levels reached 78 ± 7% of wt-CFTR (Figure 5A,C; black dotted line in Figure S3B). As to removal of RI<sup>L</sup> alone from F508del-CFTR, it restored function to 92 ± 7% of wt-CFTR (Figure 5A,C; black line in Figure S3B), as described [13]. Also, the delay in peak response observed for F508del-CFTR (min = 4) vs. wt-CFTR (min = 2), was partially corrected (to min = 3) both by removal of RI<sup>L</sup> alone from F508del-CFTR or together with RE<sup>S</sup> (Figure 5C; Figure S3B, black line and dotted line).

Introduction of G550E and R1070W revertants into the ∆RIL-F508del-CFTR variant slightly but not significantly decreased its function from 92 ± 7% to 71 ± 8% and 76 ± 8%, respectively (Figure 5A,C; black lines in Figure S3C,D), in parallel to the respective processing levels (Figure 1). In fact, these variants showed higher activity levels than the revertants containing RI<sup>L</sup> (29 ± 3% and 56 ± 6%, respectively). Interestingly however, both G550E- and R1070W-∆RIL-F508del-CFTR fully corrected (to min = 2) the delay in peak response of F508del-CFTR, which was still partially present in ∆RILand ∆RIL-RES-F508del-CFTR (min = 3).

The effect of VX-809 (3µM, 48 h at 37 ◦C) was also examined at functional level on the ∆RES-, ∆RIL-∆RES- and ∆RIL-F508del-CFTR variants. VX-809 shows a tendency to increase in <sup>∆</sup>RES-∆RIL-F508del-CFTR function (84 <sup>±</sup> 8% of wt-CFTR) which was parallel to the observed increase in PM expression (Figure 4; Figure 5A,C; black dotted line in Figure S3F). Also, ∆RES-∆RIL-F508del-CFTR fully corrected (to min = 2) the delay in peak response of F508del-CFTR which was still partially present in ∆RIL- and ∆RIL-∆RES-F508del-CFTR (min = 3).

In contrast to the observed increase in processing, VX-809 caused no significant increase in the function of ∆RIL-F508del-CFTR and actually a slight, but not significant decrease was observed and there was still the same delay in peak response (Figure 5A,C; black line in Figure S3F).

A similar slight decrease in function was observed for VX-809 on the ∆RIL-R1070W-F508del-CFTR variant (Figure 5A,C; black line in Figure S3H), but the most striking result was the significant decrease caused by VX-809 on function of <sup>∆</sup>RIL-G550E-F508del from 76 <sup>±</sup> 8% to 41 <sup>±</sup> 3% (Figure 5A; black line in Figure S3G). Nevertheless, both revertant variants of ∆RIL-F508del-CFTR showed no change in peak response which was still the same as wt-CFTR (min = 2).

Overall, VX-809 jointly with RE and RI removal completely restored F508del-CFTR function as chloride channel.

**Figure 5.** Functional characterization of the ∆RE<sup>s</sup> and ∆RI<sup>l</sup> variants of F508del-CFTR with or without VX-809 or VX-770 treatments. (**A**,**B**) Summary of the data from the iodide efflux peak magnitude generated by BHK cells stably expressing different CFTR variants and treated with 3 µM VX-809 (black bars on panel A) or with DMSO as control (white bars on panel a) for 48 h and stimulated with 10 µM Forskolin (Fsk) and 50 µM Genistein (Gen). In (**B**) cells were not pre-incubated and were stimulated either with 10 µM Fsk and 50 µM Gen (white bars on panel B) or with 10 µM Fsk and 10 µM VX-770 (black bars on panel b), as indicated. Data are shown as a percentage of wt-CFTR activity under Fsk/Gen stimulation and as mean ± SEM. (n) indicates number of independent experiments. "#" indicates significantly different (*p* < 0.05) from the same CFTR variant incubated with DMSO (A) or significantly different from the same CFTR variant under Fsk/Gen stimulation (white bars) (B). (**C**) Summary of iodide efflux data for different F508del-CFTR variants without REs, RIs, and RI<sup>l</sup> alone or jointly with revertants R1070W and G550E.
