IntroductionHuntington’s disease (HD) is a fatal neurodegenerative disease caused by the expansion of a glutamine-coding CAG repeat beyond a critical threshold in the huntingtin (HTT) gene1. The clinical course of HD is characterized by progressive affective, cognitive, and motor disturbances, notably including chorea and other movement abnormalities2. HD is inherited in an autosomal dominant manner, with disease fully penetrant beyond 39 CAG repeats3,4,5. Onset of HD symptoms generally occurs in middle age, and age of motor dysfunction onset is highly influenced by the length of the CAG repeat, though significant variability in age of onset is observed across CAG lengths6.Somatic instability (SI), a further elongation of HTT’s expanded CAG tract in somatic tissues, has emerged as a key driver of HD pathogenesis. Evidence suggests that certain cell types, particularly striatal projection neurons, are particularly vulnerable to SI7,8,9,10,11,12,13. The impact of SI in HD onset and progression is increasingly clear, thanks to large genome-wide association studies (GWAS) that identified variants in DNA mismatch repair (MMR) pathway genes that bi-directionally alter age of motor onset in HD6,14,15. Surprisingly, uninterrupted CAG repeat length also modifies age of onset -- the “CAG tract” of most human HTT alleles contain a penultimate silent CAA interruption, and loss of this interruption leads to a markedly hastened onset of HD14,16. Intriguingly, recent GWAS evidence confirms the robust impact of these onset-hastening loss of interruptions on the age of onset of HD, while contributing the novel observation that they are counterintuitively associated with significantly reduced SI, at least in blood15. However, recent human single-cell RNAseq data support a previously proposed model linking large CAG expansion to toxicity17, claiming that toxic CAG lengths are much longer than inherited CAG lengths that cause HD, perhaps as large as 150 CAGs12. Prior mouse genetic studies18,19,20 and other preclinical evidence also supports this model, as halting SI in HD knock-in mouse models with 140 CAG repeats provides dramatic protection from HD-relevant pathological signs21, whereas the same intervention is ineffective in otherwise isogenic mice with ~190-200 CAG repeats22. An important question is which HTT gene product is the critical driver of toxicity at this CAG length. One proposal is an exon-1 only short HTT transcript variant (“HTT1a”)23 and its encoded protein that avidly enters the nucleus, seeds aggregates24, and is uniquely toxic compared to other mHTT fragments25. Because the mis-splicing event that generates HTT1a is CAG size-dependent, a feasible model of toxicity is that SI drives CAG expansion in a given neuron to a length at which sufficient HTT1a is produced and the cells quickly sicken and die.As a toxic gain-of-function disorder, there is broad interest in therapeutically reducing levels of the mutant HTT gene product(s)26. The most clinically advanced effort is tominersen, an antisense oligonucleotide (ASO) developed by Ionis Pharmaceuticals and Roche that targets both HTT alleles and robustly reduced HTT levels in the cerebrospinal fluid of HD patients in a phase 1/2 study27. Unfortunately, a subsequent phase 3 clinical trial of this ASO was halted early due to worsening trends in HD symptoms in ASO-treated patients; however, a new phase 2 study of the ASO to investigate different dosing strategies is underway (ClinicalTrials.gov ID NCT05686551)28. Other therapeutic efforts to lower HTT include allele-selective ASOs targeting only mHTT by Wave Life Sciences, virally delivered micro-RNAs that reduce HTT by targeting exon-1 by uniQure (NCT04120493), and small molecule splice modulators that reduce levels of HTT transcript via selectively inducing nonsense mediated decay (NCT05358717)29. Sangamo Therapeutics has developed zinc finger transcriptional repressors (ZFPs) targeting expanded CAG repeats for allele selective silencing (now owned by Takeda Pharmaceuticals)30. There are HTT lowering clinical and preclinical trials targeting both HTT alleles (Roche, uniQure, PTC, Skyhawk) and mHTT selectively (Wave, Takeda); that are capable of engaging HTT1a by targeting exon 1 (uniQure, Alnylam, Takeda), while others target downstream (PTC, Wave, Roche, Skyhawk). In terms of therapeutic modalities, these trials include viral gene therapy (uniQure, Takeda), oligonucleotides (Wave, Roche), and small molecules (PTC, Skyhawk).We have a long-standing interest in HTT-lowering therapies31,32,33. Motivated by a concern that HTT lowering may not be sufficient to address HD pathogenesis due to ongoing SI in HTT’s CAG tract34, we set out to better understand the relationship between HTT−lowering treatments and SI in vivo. Specifically, we wanted to understand whether any Htt−lowering treatments in HD mouse models influence the progression of SI in Htt’s CAG tract. Using a range of genetic and pharmacological tools, we probed the links between Htt−lowering treatments, transcription, and SI. In general, we find DNA-targeted HTT-lowering therapies -- notably ZFPs -- consistently reduce SI, whereas those operating in the cytoplasm do not. Unexpectedly, we observe that a modified ZFP that can bind expanded CAG repeats, but not repress transcription, nevertheless robustly reduces instability, which suggests that delivery of CAG-binding DNA proteins may prove a means of developing therapies for HD by reducing SI without lowering HTT. An analagous DNA-binding protein approach could be envisioned for any repeat expansion disorder in which SI plays a role.ResultsChronic ASO treatment reduces SI and slows transcriptionIn a previous study of the impact of peripheral silencing of Htt on HD central nervous system (CNS) phenotypes35, we suppressed Htt in peripheral tissues of a knock-in HD mouse model with an inherited allele of ~116 CAG repeats (HttQ111/+) via weekly intraperitoneal administration of ASOs targeting exon 36 of mouse Htt. Because SI in the liver is as robust as the striatum, we wondered if this chronic HTT-lowering treatment might influence the progression of SI in this tissue36. We found that treatment with a pan-Htt-targeting ASO (hereafter “Htt ASO”) from 2 to 10 months of age at 50 mg/kg/week reduced hepatic HTT levels by 64% at sacrifice35; here, we observe that this is accompanied by a 37% reduction in the somatic expansion index of the HttQ111 CAG tract in the liver (Fig. 1D, E). Somatic expansion in the striatum of these mice was unaffected by peripherally administered ASOs (Fig. S1), which are excluded by the blood brain barrier37. To confirm our observations, we generated two additional cohorts sacrificed after 5.5 or 12 months of treatment (Fig. 1A) -- timepoints flanking our initial cohort’s treatment duration -- in which we see comparable knockdown of HTT protein (Fig. 1B) and total Htt mRNA (Fig. 1C), and replicate our finding that decreased HTT levels are associated with decreased somatic expansion in the liver (Fig. 1D, E). This reduction of expansion was not attributable to inherited allele sizes, which were similar across treatment groups (see “Inherited CAG Sizes” data on Dryad: https://doi.org/10.5061/dryad.0k6djhb98). Increased duration of HTT suppression was associated with greater reductions in somatic expansion, with expansion indices reduced by 50% in the liver after 12 months of treatment (Fig. 1E).Fig. 1: Chronic ASO treatment reduces somatic instability in mutant Htt’s CAG repeat and reduces Htt transcriptional rate in the livers of HttQ111/+ mice.A Overview of peripheral ASO administration mouse cohorts75. 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