Blocking somatic repeat expansion and lowering huntingtin via RNA interference synergize to prevent Huntington's disease pathogenesis in mice

Abstract

Huntington's disease (HD) is a progressive neurodegenerative disorder with no approved therapies. Two major molecular drivers-somatic expansion of inherited CAG repeats and toxic mutant HTT (mHTT) variants-lead to neuronal dysfunction. Despite multiple trials, HTT-lowering strategies have not shown meaningful clinical benefit. Using therapeutic divalent siRNAs, we assessed the long-term impact of silencing MSH3 (a key regulator of somatic expansion), HTT, or both. In Q111 HD mice (>110 CAGs), which exhibit robust expansion, mHTT inclusions, and transcriptional dysregulation by 12 months, long-term MSH3 silencing blocked expansion, reduced inclusions, and reversed gene expression changes. HTT silencing alone had limited effect, but combined MSH3/HTT targeting synergistically eliminated inclusions and restored transcriptomic profiles. Parallel treatment in wild-type mice showed no toxicity, supporting the safety of long-term intervention. These findings position somatic expansion as a promising therapeutic target and demonstrate the potential of RNAi-based co-silencing of MSH3 and HTT as a disease-modifying strategy for HD.

Fig. 1:. Divalent siRNA silence target gene expression and block somatic repeat expansion in Q111 mice.

(a) In vivo study design and treatment paradigm. 2-month-old wild-type (WT) or Q111 mice were treated with artificial CSF (aCSF), or divalent siRNA programmed with sequences targeting a non-targeting control (NTC), MSH3, HTT, or MSH3 and HTT, and euthanized at 12 months old. (b) Divalent siRNA scaffold and (c) chemical modification pattern key. (d, e) Endpoint protein levels in WT striatum: (d) WT HTT, (e) MSH3. (f-h) Endpoint protein levels in Q111 striatum: (f) WT HTT, (g) mutant HTT, (h) MSH3. (i) Representative striatum fragment analysis curves for baseline (2 months Q111 untreated) or Q111 mice (12 months old) treated with artifical CSF (aCSF), non-targeting control (NTC) siRNA, HTT siRNA, MSH3 siRNA, or MSH3+HTT siRNA combination. (j,k) Somatic instability index quantified from fragment analysis curves in (j) striatum or (k) medial cortex. WT cohorts: aCSF, n=12; NTC, n=10; MSH3 siRNA, n=12; HTT siRNA, n=12; MSH3+HTT siRNA, n=12 mice. Q111 cohorts: aCSF, n=12; NTC, n=10; MSH3 siRNA, n=10; HTT siRNA, n=9; MSH3+HTT, n=11 mice. Statistics are one-way ANOVA with Dunnett’s multiple comparisons test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Fig. 2.. Blocking expansion eliminates focal EM48+ mHTT puncta formation, enhanced mHTT puncta lowering with HTT co-lowering

(a-g) Striatal sections probed with EM48 immunohistochemistry. Representative images shown. The top panel is 40x, and the bottom panel is an area within a boxed region of interest. White arrow indicates representative EM48+ focal puncta. (a) WT aCSF (b) 2-month-old untreated Q111 (c) aCSF treated Q111 (d) NTC treated Q111 (e) MSH3 siRNA treated Q111 (f) HTT siRNA treated Q111 (g) MSH3+HTT combination siRNA treated Q111 (h) Representative histological characterization of puncta (top) or diffuse (bottom) EM48+ mHTT events (i) Quantified puncta (right) or diffuse (right) events per mouse totaled from three 40x images across three 40 μM sections (nine images counted per mouse). Each dot is average from one mouse. (j) mHTT puncta (left) or diffuse (right) quantified over each cohort. Same data as panel i, but each dot is puncta or diffuse EM48+ event from one 40x image, not averaged per mouse. (k) Percentage of focal puncta to total EM48+ events. Each dot is the average of nine slides from one mouse. (l) Percentage of diffuse EM48+ events to total EM48+ events. Each dot is the average of nine slides from one mouse. Total EM48+ events are defined as focal plus diffuse events. n=4 mice/group strained and quantified. Statistics are one-way ANOVA with Holm-Šídák's multiple comparisons test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Fig. 3.. Blocking expansion eliminates PHP1+ mHTT aggregate formation in the Q111 mouse striatum.

(a-f) Striatal immunofluorescence of PHP1 (green) co-stained with DARPP32 (purple) and DAPI (blue). Representative images of 12-month-old (a) aCSF-treated WT (b) aCSF treated Q111 (c) NTC treated Q111 (d) MSH3 siRNA treated Q111 (e) HTT siRNA treated Q111 (f) MSH3+HTT combination siRNA treated Q111 mice. Quantification of (g) average aggregate count per field of view, (h) PHP1+ area per ROI, (i) average aggregate size. n=4 mice/group imaged and quantified. Each dot summarizes events over six regions of interest, covering three striatal slices in one mouse. Statistics are one-way ANOVA with Šídák's multiple comparisons test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Fig. 4.. Blocking expansion maintains soluble mHTT levels; blocking expansion combined with lowered huntingtin reduces HTT1a aggregation.

(a) Experimental design and treatments. (b-f) Striatal HTT protein species quantified using homogenous time-resolved fluorescence detected from respective antibody donor (D) or acceptor (A) pairs across time points and treatment groups. (b) Full-length total HTT (d: MAB2166-Tb, a: CHDI-1414-d2) (c) Soluble full-length mHTT (excludes HTT1a) (d: MAB2166-Tb, a: 4C9–488) (d) Total soluble mHTT (d: 2B7-Tb, a: MW1-d2) (e) Soluble HTT1a (d: 2B7-Tb, a: 11G2-d2) (f) Aggregated HTT1a (d:4C9-Tb, a:11G2-d2). Statistics are one-way ANOVA with Dunnett’s multiple comparisons versus NTC. Mice per group at two-month timepoint: WT n=6, Q111 n=6; Mice per group at 11-month endpoint: untreated WT n=7, NTC siRNA Q111 n=8, MSH3 siRNA Q111 n=7, HTT siRNA Q111 n=7, MSH3+HTT siRNA Q111 n=6. (g) Quantified western blot probing for HTT1a using antibody 1B12 in the striatum of the cohort of Q111 mice presented in Figure 1A. Statistics are one-way ANOVA with all comparisons and Tukey’s multiple comparison test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Fig. 5.. Bulk striatum transcriptomics demonstrates blocking CAG expansion with HTT co-silencing synergies to reverse HD neurodegenerative signature beyond either treatment alone.

(a) Volcano plot of the differential gene expression analysis performed using DESeq2 between 12-month-old vehicle (aCSF-NTC) Q111 versus WT mice. Red indicates significant gene expression change. Significance determined by fold change > 1.5, FDR < 0.05. (b) Gene ontology biological processes (GOBP) pathway enrichment of the differentially expressed genes between vehicle Q111 and WT mice. (c-e) Overall gene reversal percentage of Q111 mice treated with (c) HTT siRNA (d) MSH3 siRNA or (e) MSH3+HTT siRNA combination compared to vehicle Q111 mice. Color indicates the degree of reversal classified as full, partial, super, exacerbation, or negligible. (f-i) Prevention, reversal, recuse (PuRR) classification of the degree of gene expression reversal. Number of genes differentially expressed or reversed by siRNA treatment in the (f) whole HD signature or (h) select STR266 HD signature genes across treatment cohorts as defined by Marchionini et al. 2022. (g-i) classification of the degree of reversal for the (g) whole HD signature or (i) STR266 top gene list. Degree of gene expression reversal color coded as: Blue- full reversal (F-Rev); green- partial reversal (P-Rev); orange- super reversal (S-Rev); red- exacerbation (Exac); gray- negligible reversal (N-Rev). Transcriptomics performed on the same mouse cohorts presented in Figures 1–3. N=9–12 mice per treatment group.

Fig. 6:. Time-resolved HTT, HTT1a, CAG expansion model of HD biology.

Model of HTT species across time with (a) artificial CSF or non-targeting control siRNA, (b) HTT siRNA, (c) MSH3 siRNA, (d) MSH3+HTT siRNA combination. Total full-length mutant HTT (red), soluble HTT1a (green), diffuse mHTT aggregates (blue), or mHTT inclusions (purple).