Fig. 4. RECTAS restores IKBKAP-familial dysautonomia (FD) exon 20 inclusion in patient induced pluripotent stem cells-derived sensory neurons (iPSC-SNs) and transgenic mice.

a Diagram for preparation of iPSC-SNs, showing time-course (upper), microscopic images of spheroids at day 2 (lower, left), and differentiated iPSC-SNs on day 5 (lower middle for brightfield and lower right for BRN3A/SOX10/Nucblue/TUBB3 immunocytostaining image). BF, bright field image; scale bars, 100 µm. b, c RT-PCR for IKBKAP exon 19–21 and ACTB as a loading control for differentiated iPSC-SNs from a healthy donor (WT) or FD patient (FD) on day 12 (b), and iPSC-SNs (day 12) treated with CaNDY (10 µM) or 0.1% DMSO for 2 h, followed by addition of RECTAS (10 µM) or 0.1% DMSO in media and subsequent incubation for 22 h (c). IKBKAP and ACTB were detected with primers oAM138 + oAM139 and oAM13 + oAM14, respectively. d, e Microscopic images (d) and quantification for the extent of IKBKAP-FD exon 20 inclusion (e), following treatment with RECTAS (10 µM for (d), and 2 and 10 µM for (e)) or solvent only (0.1% DMSO) for 24 h in neuro 2A cells transfected with the IKBKAP-FD reporter. Bars, 100 µm in (d). f RT-PCR for exogenous human IKBKAP (oAM124 + oAM126, primers designed for vector backbone) and endogenous mouse Ikbkap (oAM666 + oAM667, primers designed for exon 18 and 20) in neuro 2A cells transfected with IKBKAP-WT or -FD reporter and treated with 2 or 10 µM RECTAS or 0.1% DMSO for 24 h. Actb (oAM364 + oAM366) served as a loading control. g Quantification of PSI for IKBKAP-FD exon 20 in DRG from IKBKAP-FD-humanized transgenic mice administered RECTAS (300, or 400 mg/kg BW, p.o.) with a booster administration of the same dose at 4 h, and DRG were collected 8 h after the first administration to be applied for RT-PCR for IKBKAP exon 19–21 with primer set of HsIKAPRT18F + HsIKAPRT21R (n = 2 for no treatment, n = 6 for MOCK, n = 5 for 300 mg/kg BW RECTAS, and n = 3 for 400 mg/kg BW RECTAS). MOCK, control for the vehicle only. h RT-PCR for Rbm24 in blood cells, skeletal muscle, and liver tissues from 2-month-old B6 mouse, as well as DRG from MOCK or RECTAS (300 mg/kg BW)-administered IKBKAP-FD transgenic mice. Actb served as a loading control. Rbm24 and Actb were detected by primers Rbm24-F + Rbm24-R and oAM365 + oAM366, respectively. Data from three iPSC clones are shown in (a–c), four replicates were shown in (d–e), two replicates are shown in (f). Representative data from two experiments are shown in (h). E20 (+), exon 20 inclusion product; E20 (−), exon 20 skipping product; E19 (+), exon 19 inclusion product; E19 (−), exon 19 skipping product in (b), (c), and (f). Columns, mean; bars, SE; n.s., P ≥ 0.05; **P < 0.01; ***P < 0.001 for unpaired two-tailed t test in (e) (vs. DMSO control: 2 µM, P = 5.6 × 10⁻⁵; 10 µM, P = 1.5 × 10⁻⁴) and (g) (vs. MOCK control: no treat, P = 0.97; 300 mg/kg BW, 2.4 × 10⁻³; 400 mg/kg BW, 1.0 × 10⁻²); PSI, percent spliced-in in (b), (c), and (f); ND, not detectable in (f).