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.