F508del/F508del intestinal organoids respond positively to RDR01752
Next, we tested the effects of RDR01752 using the FIS assay in intestinal organoids with the F508del/F508del genotype. Organoids were incubated with RDR01752, VX-809 or VX-661 for 24h and then acutely stimulated (30 min) with Fsk with or without a potentiator (VX-770 or Gen) to further enhance CFTR function (Figs. 3 and S1). Significant swelling was observed in organoids incubated with RDR01752, VX-809 or VX-661 and acutely stimulated with either VX-770 or Gen, in contrast to absence of swelling in organoids without any potentiator or DMSO control. Interestingly, similar swelling values were observed for organoids incubated with any corrector plus potentiator combination.
RDR01752 increases the rescue of F508del-CFTR PM in cells expressing in cis the genetic revertants G550E and 4RK or low temperature, but not in R1070W nor DD/AA
In order to characterize the mechanism of action (MoA) by which RDR01752 rescues F508del-CFTR, we investigated revertants of this mutant. To this end, CFBE cell lines stably expressing double tagged-F508del-CFTR incis with the following genetic revertants: G550E, R1070W, or 4RK were incubated with this compound. In parallel CFBE cells expressing WT-CFTR or the traffic-null variant DD/AA (on a WT background) were also treated with RDR01752. Cells were incubated both at 37ºC and low temperature (27ºC), and PM expression of each CFTR variant was assessed by immunofluorescence (Fig. 4A,B, respectively) and normalized to cells incubated with DMSO at 37°C (upper heatmap, first row).
Each of the correctors tested RDR01752, VX-809 or VX-661 rescued F508del-CFTR to the PM with similar efficacy (Fig. 4A) which was further enhanced when incubated at 27°C (Fig. 4B). These compounds also increased WT-CFTR PM expression, although VX-809 and VX-661 showed higher efficacy than RDR01752. A small additive effect was found for PM levels of wt-CFTR with each of these compounds and low temperature.
Analysis of the effects on the revertants demonstrated that RDR01752, similarly to VX-809 and VX-661, is additive to G550E and 4RK in F508del-CFTR PM rescue (Fig.4A), and further additive to that of low temperature (Fig. 4B). In contrast, RDR01752, also similarly to VX-809 and VX-661, did not demonstrate additivity with R1070W (Fig. 4A), thus suggesting that, like VX-809 (Farinha et al., 2013), it might share a common mechanism. Furthermore, an increase in PM expression of DD/AA-CFTR variant was not observed (Fig. 4A), except when cells were incubated at low temperature (Fig. 4B), as before (Farinha et al., 2013), with no significant additive effect elicited by any of the correctors.