*2.4.* ∆*RI<sup>L</sup> Synergises with VX-809, but Not with Revertants to Rescue* ∆*RES-F508del-CFTR Processing*

To further test how the stabilized immature form of ∆RES-F508del-CFTR could be pharmacologically rescued, we then assessed the impact of corrector VX-809 (which per se promotes maturation of the F508del-CFTR) on the processing of this and of the other variants, to obtain structural insight on the effects of this novel drug. Although ∆RES-F508del-CFTR could not be rescued by VX-809, data show that this small molecule was able to rescue <sup>∆</sup>RIS-∆RES-F508del-CFTR (from 7 <sup>±</sup> 2% to 20 <sup>±</sup> 4%) and further increased processing of <sup>∆</sup>RIL-∆RES-F508del-CFTR to wt-CFTR levels (from 71 <sup>±</sup> 3% to 96 <sup>±</sup> 2%) (Figure 3A, lanes 6,7; Figure 3E; Table 1). These data suggest a strong synergistic effect between VX-809 and ∆RI<sup>L</sup> (and with ∆RIS, albeit to a lesser extent) to rescue ∆RES-F508del-CFTR processing. This is particularly interesting because VX-809 did not rescue processing of the ∆RES-, nor ∆RIS-F508del-CFTR variants (Figure 3A, lanes 5,3; Table 1) while it recovered the processing of ∆RIL-F508del-CFTR to wt-CFTR levels (Figure 3A, lane4; Table 1). Notably, VX-809 was able to almost completely revert processing impairment of <sup>∆</sup>RIS-wt-CFTR and <sup>∆</sup>RIS-∆RES-CFTR (from 44 <sup>±</sup> 2% and 54 <sup>±</sup> 9%) to 90 <sup>±</sup> 3% and 93 ± 3%, respectively (Figure 3A, lanes 8,11; Figure 3D; Table 1).

Strikingly, VX-809 had no effect on processing of ∆RES-F508del-CFTR with R1070W or G550E (Figure 3C, lanes 6,10; Figure 3E; Table 1), while it further rescued processing of F508del-CFTR variants with R1070W or G550E alone to 46 ± 4% and 73 ± 6%, respectively (Figure 3C, lanes 3,7; Figure 3E; Table 1), as reported [5]. Similarly, VX-809 caused no significant further rescue on the processing of ∆RIL-F508del-CFTR with those revertants, but these variants already had processing levels close to those of wt-CFTR (Figure 3C, lanes 5,9; Figure 3E; Table 1). Interestingly, the impaired processing of <sup>∆</sup>RES-R1070W-wt-CFTR (71 <sup>±</sup> 6%) was also reverted by VX-809 to 90 <sup>±</sup> 3% (Figure 3B, lane 6; Table 1).

Corrector VX-809 failed to rescue any of the ∆REL-, ∆H9-, and ∆REL-∆H9-F508del-CFTR variants (Figure 2B,C), while significantly increasing the processing of the <sup>∆</sup>REL-∆H9-wt-CFTR from 55 <sup>±</sup> 3% to 69 ± 3% (Figure 2B, lane 7; Figure 2C).

Altogether, these results suggest that RI<sup>L</sup> and the genetic revertants interfere with the rescue of F508del-CFTR by VX-809.
