Fig 2: Selectivity of compound-induced splicing.a Volcano plot of RNA-Seq analysis comparing gene expression in SH-SY5Y cells treated with either 24 nM or 100 nM of HTT-C2 with dimethyl sulphoxide (DMSO) treatment. mRNAs with significant changes in expression (>1.5-fold, false discovery rate (FDR) < 5%) are shown as blue and red dots for down- and upregulation, respectively. b A schematic of alternative splicing (AS) events. CE, cassette exon; A3SS, alternative 3' splice sites (ss); A5SS, alternative 5'ss. c Number of regulated AS events in SH-SY5Y RNA-Seq data following treatment with 24 nM and 100 nM HTT-C2. d Number of CEs inclusion (Inc) or skipping (Skp) after HTT-C2 treatment; ratio of Inc/Skp are shown in text. e Percentage of exons with 3' and 5'ss annotated by public databases (Refseq, Ensembl, or UCSC Known Genes) for NC (no change) or Inc exons. f Cumulative distribution function (CDF) curves of basal percent-spliced-in (PSI) index (average PSI in DMSO samples). The graph shows data for exons separated into three groups; Inc is based on ?PSI > 20% and two-sided Fisher’s Exact Test P < 0.001 in any one of the two conditions (24 nM or 100 nM HTT-C2 vs. DMSO). Median values are shown as dashed vertical lines for each group. g Sequence conservation of 3'ss and 5'ss region. Conservation is based on phastCons score for 46-way placental mammals. Mean (standard error of mean [SEM]) conservation scores are shown. h CDF curves of RNA-Seq mRNA abundance change for genes with predicted nonsense-mediated mRNA decay (NMD)-psiExons. NMD-psiExons are psiExons whose inclusion in mRNA introduces a premature termination codon or causes frameshift or both, and are included (Inc) following HTT-C2 treatment. Number of genes (n) and P value are indicated. P value is based on comparison with “all other genes” group using Wilcoxon rank-sum test (two-sided).

Fig 3: SIP protein expression is dysregulated in the striatum in YAC128 mice. a Detection of SIP monomer and dimer in protein extracts from the striatum in YAC128 mice (YAC128) vs. control mice (WT) using Western blot (WB) under denaturing conditions. SIP densitometry was calibrated based on the intensity of p-cadherin. m, SIP monomer; d, SIP dimer. b Increase in levels of SIP dimers in YAC128 mice vs. control mice presented as fold changes; **p < 0.01. c Protein levels of SIP monomers in YAC128 mice vs. control mice presented as fold changes. ns not significant. d Detection of SIP monomer in protein extracts from HEK293T cells. m, SIP monomer. e Interaction between SIP and huntingtin (HTT). Samples that were subjected to immunoprecipitation (IP) with anti-SIP antibody were obtained from the striata in YAC128 mice and control mice (WT). Blots were labeled using an anti-HTT antibody. Western blot analyses of total extracts from YAC128 mice were used as controls (Total). Ab, anti-SIP antibody used for IP; M, protein marker in kilodaltons (kDa); IgG, heavy chain of antibody. The results are expressed as mean ± SEM. The results were obtained from the striata of two or four independent YAC128 and control mice for the IP and WB experiments, respectively

Fig 4: Regulation of compound-induced splicing for HTT-C2 and SMN-C3.a 5' splice sites (ss) sequence at regions (-4 to -1 and +1 to +6) were studied for the enrichment of Inc (included) vs. NC (no change) exons. Significance scores are shown. Wide boxes represent exons. b Schematic of 5'ss sequence logo in the three exon groups: annotated NC, annotated (inclusion) Inc and psiExons Inc. c Diagram illustrating the design of huntingtin (HTT) minigene constructs for studying compound-induced splicing. d Polymerase chain reaction (PCR) analysis of RNA extracts from HEK293 cells transfected with wild-type (wt) human HTT minigene or constructs with point mutations in the -2 to +3 region of the 5'ss; cells were treated with dimethyl sulphoxide (DMSO) or HTT-C2 (0.010–1 µM). The data are from a single transfection experiment with multiple concentrations tested for a given construct; the “WT” control construct has been used multiple times (n > 3). e Sequence of the 20-nucleotide region upstream of the 5'ss of HTT stop-codon psiExon49a showing partial deletions and mutations performed on this region, and their effects on HTT-C2 induced splicing (lower panel). Through partial deletion or mutation of the nucleotides CAGGA at positions -38 to -34, this region was shown to be important in regulating splicing events. The data are from a single transfection experiment with multiple concentrations tested for a given construct; the “WT” control construct has been used multiple times (n > 3).

Fig 5: Huntingtin (HTT)-lowering activity in vitro.a Chemical structures of HTT-C1 and HTT-D1. b Electrochemiluminescence (ECL) analysis of mutant HTT protein from fibroblasts isolated from a homozygous patient with Huntington’s disease (HD) (GM04857) after 96 h of continuous treatment with HTT-C1 and HTT-D1 (0.01–1.0 µM). Representative graphs show percent HTT remaining relative to the dimethyl sulphoxide (DMSO) control. Cell viability assays were performed in parallel. Data represent mean of two (n = 2) biologically independent samples per data point from one dose–response experiment. c Western blot of HTT protein and housekeeping proteins, oxidoreductase-protein disulphide isomerase (PDI), beta-actin, alpha serine/threonine-protein kinase (AKT) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in HD fibroblasts after 96 h of continuous treatment with HTT-C1 (0.015–1.0 µM). Utrophin (UTRN) was also used as a loading control. The western blot data used a representative splicing modifier (tested at multiple concentrations) from a single experiment. Multiple splicing modifiers from the same class were tested and evaluated by western blot analyses. d Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis of HTT mRNA in patient fibroblasts after 24 h of treatment with HTT-C1 and HTT-D1 (0.01–1.0 µM). Representative graphs show percent HTT mRNA remaining relative to DMSO control; normalised to housekeeping gene, TATA-box binding protein. Data represent mean of two (n = 2) biologically independent samples per data point from one dose–response experiment.