Fig. 7: Disrupting PTBP2 binding in Site 1 and Site 2 upregulates SYNGAP1 mRNA in SYNGAP1 haploinsufficient patient cell lines.
From: Mapping PTBP2 binding in human brain identifies SYNGAP1 as a target for therapeutic splice switching
a Schematic of SYNGAP1 mRNA showing the location of the heterozygous mutations present in the two independent SYNGAP1 patient iPSC lines. b Schematic depicting the generation of the SYNGAP1 R1240X patient-derived iPSC line and the corresponding isogenic control line in which the heterozygous mutation has been reverted using CRISPR/Cas9 technology. PBMCs peripheral blood mononuclear cells. Created with BioRender.com. c Top and right panels: SYNGAP1 Western blot from corrected (isogenic control) and patient SYNGAP1 R1240X iPSC-neurons. d Top and right panels: SYNGAP1 Western blot from WT and patient SYNGAP1 K1185X iPSC-neurons. c, d Left panel: qPCR showing SYNGAP1 mRNA levels. e Top and right panels: PTBP2 Western blot from K1185X NPCs treated for 3 d with PTBP2 gapmer. Left panel: PTBP2 mRNA fold change quantification (qPCR). f Top and left panels: SYNGAP1 RT-PCR from K1185X NPCs treated for 3 d with PTBP2 gapmer. Right panel: qPCR showing SYNGAP1 mRNA levels. g SYNGAP1 Western blot from samples in e. e–g a non-targeting gapmer (NegA) was used as negative control. h Top and left panels: RT-PCR from R1240X iPSC-neurons treated for 7 d with ET-019 at 10 µM. Right panel: qPCR showing SYNGAP1 mRNA levels. i Top and left panels: RT-PCR from K1185X iPSC-neurons treated for 7 d with Site 1 and Site 2 targeting ASOs at 10 µM. Right panel: qPCR showing SYNGAP1 mRNA levels. White color data points represent an independent experiment. h, i Non-targeting ET-SC or ET-MM ASOs were included as negative controls. Data are represented as mean values ± SEM. All data points represent independent biological replicates. c, d (n = 3). e–h (n = 6). i (n = 3 except n = 5 for Mock and n = 6 for ET-019 and ET-020). c, d Student’s t test. In e–g, one-way ANOVA with Dunnett’s multiple comparison test vs. NegA-treated cells. h, i One-way ANOVA with Dunnett’s multiple comparison test vs. mock-treated cells. Source data are provided as a Source Data file. FC fold change.
