Splice modulators target PMS1 to reduce somatic expansion of the Huntington's disease-associated CAG repeat

Abstract

Huntington's disease (HD) is a dominant neurological disorder caused by an expanded HTT exon 1 CAG repeat that lengthens huntingtin's polyglutamine tract. Lowering mutant huntingtin has been proposed for treating HD, but genetic modifiers implicate somatic CAG repeat expansion as the driver of onset. We find that branaplam and risdiplam, small molecule splice modulators that lower huntingtin by promoting HTT pseudoexon inclusion, also decrease expansion of an unstable HTT exon 1 CAG repeat in an engineered cell model. Targeted CRISPR-Cas9 editing shows this effect is not due to huntingtin lowering, pointing instead to pseudoexon inclusion in PMS1. Homozygous but not heterozygous inactivation of PMS1 also reduces CAG repeat expansion, supporting PMS1 as a genetic modifier of HD and a potential target for therapeutic intervention. Although splice modulation provides one strategy, genome-wide transcriptomics also emphasize consideration of cell-type specific effects and polymorphic variation at both target and off-target sites.

Fig. 1. Branaplam and risdiplam treatment of HD LCLs produced two major HTT alternative splice products.

a Schematic diagram showing the alternative HTT splice products upon drug treatment. b PCR from exon 49–50 showing the size of the splice products from a single experiment. c Branaplam and risdiplam dose response for each HTT splice product. d Quantification of splice products produced from mutant minigenes following transfection of HEK 293 T cells either treated with a vehicle control (DMSO) or 100 nM branaplam. Source data are provided as a Source Data file.

Fig. 2. Two single nucleotide variants affected HTT splice modulation.

a Minor allele frequency (MAF) of variants spanning HTT exon 49–50 (exons marked with solid vertical lines), with variants represented in the cell lines tested labeled and highlighted in blue. The dotted vertical lines indicate the pseudoexon splice sites (ss). b The proportion of canonical HTT exon 49–50 product across tested cells lines, grouped by heterozygous presence of variant. Since the production of the pseudoexon requires drug treatment, only a subset of the cell lines were treated with DMSO control. c Absolute quantification by ddPCR across exon 49–50 junction for a subset of the cell lines on a log10 axis. Box plots show the 25th and 75th percentiles (box), median (horizontal line), and range (whiskers, which are capped at 1.5x the inter-quartile range). N = Number of cell clones, n = cultures analyzed. Source data are provided as a Source Data file.

Fig. 3. RPE1-AAVS1-CAG115 cell model for CAG repeat instability.

a Schematic diagram of the key elements of transgene integration in the AAVS1 site in intron 1 of PPP1R12C. The PPP1R12C promoter (blue) drives transcription from exon 1 to 2 of the endogenous gene (gray), which was analyzed with allele specific primers due to a polymorphism in exon 2 (orange line). The puromycin resistance gene (PuroR, yellow) is driven from the same promoter with a new 3’splice site (3’SS, yellow). HTT exon 1 / EGFP expression (both in yellow with location of the CAG repeat shown as a partial sequence) is driven in the opposite direction from tetracycline-responsive element (TRE) promoter (blue). b RPE1-AAVS1-CAG115 brightfield images of cells nearing confluency (Day 0) and contact inhibited at confluency for four days (Day 4) (scale bar 0.1 mm). c Quantification of the number of RPE1-AAVS1-CAG115 cells in S-phase by image analysis. Edu-positive nuclei represent cells that incorporated Edu into their DNA and stained positive upon labelling, which was normalized on the total number of nuclei per field of view (160 images analyzed per group). Box plots show the 25th and 75th percentiles (box), median (horizontal line), and range (whiskers, which are capped at 1.5x the inter-quartile range). d CAG repeat fragment distribution for a single RPE1-AAVS1-CAG115 clone in the absence (blue, middle) or presence (red, bottom) of doxycycline-induced transcription compared to day 0 (top). (e) CAG repeat fragment distribution clone in the absence (blue) or presence (red) of doxycycline-induced transcription for non-dividing (middle) and dividing (bottom), compared to day 0 (top). f Average repeat gain in non-dividing or dividing cells in a single RPE1-AAVS1-CAG115 clone with either non-induced (blue) or induced (red) transcription. The n indicates the number of cultures analyzed. g GFP fluorescence signal analyzed by flow cytometry in parental RPE1 (no transgene) and RPE1-AAVS1-CAG115 cells with either non-induced (blue) or induced (red) transcription. h Relative expression, normalized to expression of reference gene SDHA, of PPP1R12C exon 1–2 (allele specific from the transgene chromosome), PuroR (specific to transgene with assay designed to be unable to detect puromycin in parental RPE1 from hTERT transgene) and EGFP by ddPCR, with either non-induced (blue) or induced (red) transcription. Three replicates analyzed for each condition and data displayed on a log10 transformed axis. Source data are provided as a Source Data file.

Fig. 4. RPE1-AAVS1-CAG115 cell model repeat expansion depends on known modifiers of HD onset and CAG repeat instability.

a Fragment analysis traces showing the change in CAG repeat length distribution across time in different non-edited and edited cells for pooled edited populations. Color indicates CRISPR-Cas9 knock-out (KO): non-targeting empty vector (black), FAN1 (purple), MSH3 (red), and PMS1 (orange). The plots represent raw fluorescent signal without baseline correction and therefore have a negative signal bias with increasing fragment size. The following instability metrics were derived from data processed in the GeneMapper software which corrects this bias. b Average repeat gain for pooled edited populations, with each dot representing a biological replicate. c Average repeat gain for cell clones isolated from either MSH3 (red) or PMS1 (orange) targeted populations. Box plots show the 25th and 75th percentiles (box), median (horizontal line), and range (whiskers, which are capped at 1.5x the inter-quartile range). N = Number of cell clones, n = cultures analyzed. Source data are provided as a Source Data file.

Fig. 5. Branaplam and risdiplam treatments reduced repeat expansion in RPE1-AAVS1-CAG115 cells.

Average repeat gain of non-induced RPE1-AAVS1-CAG115 cells with treatment of either branaplam (a) or risdiplam (b), with the color indicating the drug concentration. Each treatment group and timepoint had five cultures analyzed, except risdiplam day 0 which had three. c Drug cytotoxicity quantified by high-throughput image analysis of cells treated with DNA labeling of dead cells. d Average background autofluorescence pixel intensity. For c and d, 81 images were analyzed per treatment. Box plots show the 25th and 75th percentiles (box), median (horizontal line), and range (whiskers, which are capped at 1.5x the inter-quartile range). Source data are provided as a Source Data file.

Fig. 6. HD modifier PMS1 contains a drug-inducible pseudoexon.

a Schematic diagram of the PMS1 transcript (NM_000534) highlighting the pseudoexon location in red. b Dose response of PMS1 exon 5–6 (canonical band) after branaplam (teal) or risdiplam (red) treatment with each empty dot representing a biological replicate, the line showing the local polynomial regression, and the ribbon displaying the standard error. c Western blot showing decreased PMS1 protein in RPE1-AAVS1-CAG115 cells after branaplam or risdiplam treatment for 10 days with DMSO or the indicated drug concentrations predicted from the RNA dose response to represent IC50, IC75, and IC90. The +/+ and -/- lanes represent non-edited wild-type and edited PMS1 knock-out RPE1 cells, respectively. The samples are from a single experiment with three biologically independent samples per treatment. Source data are provided as a Source Data file.

Fig. 7. PMS1 pseudoexon inclusion explained the effect on CAG repeat expansion with branaplam, but only partially with risdiplam.

a Schematic diagrams showing the CRISPR-Cas9 targeting approach for the disruption of pseudoexon (PE) sequences in HTT (left) and PMS1 (right). Yellow indicates pseudoexon sequence upstream of the wild-type (WT) 5’ splice site targeted by the drug, blue representing the downstream intronic sequence, with the PE mutation (ΔPE) sequence highlighted in purple. b PCR analysis over the HTT (top) and PMS1 (bottom) pseudoexon splice junctions with branaplam or risdiplam treatment for the control and pseudoexon edited cell lines. The data are from a single experiment. c Accurate quantification of PMS1 canonical isoform by ddPCR for the control and PMS1 pseudoexon edited cell lines. Each unique cell line is represented by a different dot color. d, e The average repeat gain per week after branaplam or risdiplam treatment for the different edited cell lines (dot color), normalized on the average repeat gain in the DMSO for each genotype. Box plots show the 25th and 75th percentiles (box), median (horizontal line), and range (whiskers, which are capped at 1.5x the inter-quartile range). Source data are provided as a Source Data file.

Fig. 8. SpliceAI identified variants predicted to affect splicing of branaplam-responsive exons genome-wide.

a The overlap of genes with drug-responsive exons in the different studies analyzed for branaplam and risdiplam treatment. For each comparison, the number of genes in common across each dataset (white number) was normalized on the total number of genes for each row (black number). The color indicates the overlap proportion (white number / black number). b SpliceAI predictions were made for variants within 50 nt of branaplam-responsive exon and pseudoexon splice junctions. c Variants near branaplam- (left) or risdiplam-responsive pseudoexons (orange) and exons (green) that yield significant SpliceAI scores are plotted by allele frequency with gene names indicated for selected variants. HTT variants rs148430407 (MAF 2.6×10⁻³) and rs772437678 (MAF 9.6×10⁻⁵) are labelled, while rs145498084 did not produce a significant score. (d) SpliceAI-predicted variants affect splice modulation of TENT2 and ZFP82. Proportion of canonically spliced product across tested LCLs for TENT2 and ZFP82, grouped by absence (0/0) or heterozygous presence (0/1) of variant. N = Number of cell lines for variant, n = cultures analyzed. Source data are provided as a Source Data file.