Suppression of Huntington's Disease Somatic Instability by Transcriptional Repression and Direct CAG Repeat Binding

Abstract

Huntington's disease arises from a CAG expansion in the huntingtin gene beyond a critical threshold. Current therapeutics primarily aim to reduce toxicity by lowering levels of mutant HTT mRNA and protein. Genetic data support a role for somatic instability in HTT's CAG repeat as a driver of age of motor dysfunction onset, but currently, the relationship between instability and HTT lowering remains unexplored. Here, we investigate various HTT-lowering modalities to establish the relationship between HTT lowering and instability in Huntington's disease knock-in mice. We find that repressing transcription of mutant Htt reduces instability, using genetic and pharmacological approaches. Remarkably, zinc finger proteins that target CAG repeats, but lack a repressive domain, protect from somatic instability despite not reducing HTT mRNA or protein levels. These results suggest that DNA-targeted HTT-lowering treatments may have advantages compared to other HTT-lowering approaches, and that steric blockage of CAG repeats may reduce instability while sparing HTT expression.

Fig. 1. Chronic ASO treatment reduces somatic instability in mutant Htt’s CAG repeat and reduces Htt transcriptional rate in the livers of Htt^Q111/+ mice.

A Overview of peripheral ASO administration mouse cohorts. B Chronic ASO treatment reduces levels of both wildtype (63% reduction; p = 0.001) and mutant HTT (54% reduction; p = 0.086) in the liver, as assayed with MSD at 14 months of age following 12 months of ASO treatment. C Total Htt mRNA, as assayed by qRT-PCR, is also reduced by chronic ASO treatment (94% reduction; p = 0.0008). D Exemplar traces of the size distribution of PCR products from a CAG-spanning PCR reaction. The top panel arises from a mouse treated with saline, while the bottom one is treated with Htt-targeted ASO. E Somatic instability is reduced by chronic peripheral dosing of Htt ASO, regardless of length of treatment (39%, 35%, 51% reductions at 7.5, 10 and 14 months, respectively; p = 0.0002, p = 1.29e-10, p < 5e-19). F Chronic ASO treatment reduces the levels of Htt pre-mRNA, as quantified by qRT-PCR with primer pairs that span the indicated exon-intron boundaries, with reduction increasing downstream of the ASO binding site (exon 2-intron 2: 44% reduction, p = 0.035; intron 37-exon 38: 66%, p = 0.009; exon 66-intron 66: 69%, p = 0.002). G Overview of locations of the ASO target and exon/intron primer pairs. For box and whisker plots, each data point is shown, and the horizontal lines indicate the 25th, 50th and 75th percentiles of the data, with vertical lines indicating the range, with outliers detached from the vertical lines. Cohort 1: n = 3-6 mice/arm; cohort 2: n = 8-9 mice/arm; cohort 3: n = 5-6 mice/arm. All p-values reported are results of Tukey’s HSD test.

Fig. 2. Partial lac repressor-mediated transcriptional repression of mHtt reduces somatic instability in Htt’s CAG repeat in the livers of Htt^Q140 mice.

A Overview of the mechanisms of action of the lac repressor system. B Schematic of study design indicating when mice in 3 cohorts were treated with IPTG, reducing LacR-mediated repression of the Htt^Q140 allele. C Protein knockdown at sacrifice, measured by MSD: wtHTT is unaffected by the lac repressor (p = 0.32), while mHTT levels are reduced (46%, p = 5.48e-05). D Knockdown of total Htt mRNA from liver from replicate mouse experiment (27%, p = 0.018; NB, only mHtt is repressed, so this non-allele selective mRNA assay represents an under-estimate of mHtt-selective knockdown). E Somatic instability is reduced via partial LacR-mediated repression of mHtt, with greater suppression of instability given longer suppression durations. F Exemplar traces of repressed and unrepressed mice. For box and whisker plots, each data point is shown, and the horizontal lines indicate the 25th, 50th and 75th percentiles of the data, with vertical lines indicating the range, with outliers detached from the vertical lines. n = 8-10 mice/arm.

Fig. 3. CRISPR/Cas9-mediated deletion of the mHtt promoter region reduces SI in the Htt CAG repeat tract.

A Cartoon indicating the location of the gRNA pair referred to as “QPro,” spanning an ~1200 bp region, that targets unique sequences in the humanized Htt^Q111 allele that are not present in mouse Htt. B AAV8-delivered treatment with QPro results in robust, allele-selective lowering of HTT protein in the livers of Htt^Q111+; Cas9 mice. C Treatment with AAV8::QPro at 2.5 months of age results in reduction of expansion index at 8 months of age. D Exemplar traces of CAG tract expansions in untreated and treated animals. For box and whisker plots, each data point is shown, and the horizontal lines indicate the 25th, 50th and 75th percentiles of the data, with vertical lines indicating the range, with outliers detached from the vertical lines. n = 6-9 mice/arm.

Fig. 4. siRNA-mediated repression of Htt does not alter its somatic instability or transcriptional rate in Htt^Q111/+ mice.

A Schematic of study design. B Chronic (10 mg/kg/month) siRNA treatment reduces levels of both wildtype (74% reduction; p = 2.47e-08) and mutant HTT (55% reduction; p = 1.84e-08) in the liver, as assayed with MSD at 6 months of age following 3.5 months of treatment. C Total Htt mRNA, as assayed by qRT-PCR, is also reduced by siRNA treatment (64% reduction; p = 1.31e-06). D Htt pre-mRNA levels are not impacted by siRNA treatment (NS). E Chronic siRNA treatment does not change SI in HTT’s CAG tract (NS). For box and whisker plots, each data point is shown, and the horizontal lines indicate the 25th, 50th and 75th percentiles of the data, with vertical lines indicating the range, with outliers detached from the vertical lines. n = 10 mice/arm.

Fig. 5. Genetic loss of wtHTT does not influence somatic instability.

A, B Western blot demonstrating the lack of wildtype HTT in Htt^Q111/- striatum, and no alteration in mHTT levels. C Instability in Htt’s CAG repeat is not altered in Htt^Q111/- mice compared to Htt^Q111/+ mice in either the striatum or liver. For box and whisker plots, each data point is shown, and the horizontal lines indicate the 25th, 50th and 75th percentiles of the data, with vertical lines indicating the range, with outliers detached from the vertical lines. n = 3−11 mice/arm.

Fig. 6. ZFP treatment reduces Htt’s transcriptional rate and blocks CAG SI, while DBD-only lowers SI but spares transcription.

A Schematic of ZFP constructs: the DNA-binding domain (DBD) binds CAG repeats, while the KRAB domain induces repressive chromatin. B Schematic of treatment paradigm -- each subject mouse is treated with two viruses, one in each hemisphere. C Htt transcript levels as measured by bDNA assay decrease after ZFP treatment (full ZFP vs KRAB-only, 20% reduction, p = 0.0002) but are unaffected by DBD-only treatment (NS). NB, this is a non-allele selective assay, and so likely underestimates the amount of allele-selective lowering of mHtt. D Treatment with the complete ZFP constructs leads to a marked reduction in exon 1 only mHtt (Htt1a) levels as measured by bDNA assay (50% reduction, p = 8.05e-10), but are unaffected by DBD-only treatment (NS). E Exemplar traces from each treatment group. F Somatic instability of mHtt’s CAG tracts is reduced (70%, p < 5e-19) by the full ZFP construct, with the DBD-only construct resulting in an intermediate reduction (42%, p < 5e-19). G Results of in vitro study in STHdhQ111cells showing no reduction of transcription in DBD-only construct, measured by pre-mRNA qPCR at two locations. H mHTT protein levels measured by MSD in Htt.Q175 neurons showing no reduction of mHTT protein in DBD-only construct. For box and whisker plots, each data point is shown, and the horizontal lines indicate the 25th, 50th and 75th percentiles of the data, with vertical lines indicating the range, with outliers detached from the vertical lines. B−D, F n = 12–24 mice/arm; G n = 6 biological replicates/arm; H n = 4 biological replicates/arm.