Fig 2: Generation and validation of HttKO clones in SH-SY5Y cells. A A diagram depicting the exons of human Htt and four gRNA sequences. The structural map of Htt is generated from the UCSC Genome Browser. Exon 8 is enlarged in the box with the coded amino acid sequence at the bottom. Red sequence indicates the gRNA2 sequence. B Estimation of mutation frequencies by mismatch cleavage assay. Forty-eight hours after electroporation, genomic DNA from different gRNA- transfected cells was extracted and the fragment around the gRNA targeted sequence was amplified by PCR, cleaved by mismatch assay and analyzed by DNA gel electrophoresis. Upper panel: PCR products before re-annealing and cleavage. Lower panel: PCR products after mismatch cleavage assay. C Sanger sequencing of WT, B7 and C12 clones to identify InDels. D Representative confocal images showing the absence of Htt expression in B7 and C12 clones. All images were taken with the same laser intensity and gain. Not further adjustments were applied. Cells were immunostained with rabbit monoclonal Htt antibodies (D7F7). Nuclei were counterstained with DAPI. Scale bar: 20 µm. E Western blot analysis of Htt expression in WT SH-SY5Y cells, B7 and C12 HttKO clones. Rabbit monoclonal Htt antibody (D7F7) and mouse monoclonal ß-actin antibody were used in the blot

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: SIP increases autophagy in cells that express N-terminal mutant HTT but not in the presence of N-terminal wt HTT. a HEK293T cells were co-transfected with plasmids (control pEntry and e1HTT-25Q-RFP; wt SIP and e1HTT-25Q-RFP; control pEntry and e1HTT-72Q-RFP; wt SIP and e1HTT-72Q-RFP), pretreated with rapamycin and chloroquine, and imaged for autophagy using the Cyto-ID Autophagy detection kit. Signals from autophagosomes were detected in green. Signals that corresponded to the expression of proteins that were encoded by e1HTT-25Q-RFP and e1HTT-72Q-RFP were detected in red and yellow, respectively. Aggregates of e1HTT-72Q were imaged in yellow (white arrows) because of their high fluorescence intensity, which was detected in the FITC channel. Scale bar = 1 µM. b Area of autophagosomes in double-transfected HEK293T cells using the above plasmids. The area of autophagosomes was measured as the number of pixels per e1HTT expressed cell

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.