21 Feb 2025

At birth, people who carry a string of more than 40 CAG repeats within the first exon of the huntingtin gene are all but destined to develop Huntington’s disease. Yet, recent studies are converging on the idea that the number of inherited repeats is not always sufficient to manifest the disease. Rather, it is their subsequent expansion within subsets of neurons, some of which ultimately harbor hundreds of repeats, that ignites the devastating neurodegenerative disorder. Therein lies an opportunity to stop it, according to scientists led by Sarah Tabrizi of University College London. In the February 12 Science Translational Medicine, they reported that taking down MSH3, a DNA repair protein, nipped CAG expansion in the bud.

In stem-cell-derived striatal neurons from a person with HD, an antisense oligonucleotide did the trick. MSH3 unwittingly fosters growth of repeat sequences, and the findings suggest that reining in the somatic proliferation of CAG repeats could stave off the disease among carriers of this mutation. To move forward with preclinical studies, the scientists generated a human MSH3 knock-in mouse model. Their ASOs took down the protein throughout the mouse brain.

Previous studies have established that longer CAG expansions lead to earlier age of symptom onset in people with HD, and that somatic expansion of the repeat sequence in neurons plays a role in this acceleration (Hong et al., 2021). Tabrizi and others reported that expansion beyond 150 repeats, particularly in striatal neurons most vulnerable to the disease, heralds the onset of cell degeneration (Scahill et al., 2025; Handsaker et al., 2025; Bates, 2025). Scientists have pegged DNA repair enzymes as expanders of the repeat tract, and identified variants in the genes encoding these enzymes as modifiers of onset age.

This includes MSH3 (Gusella et al., 2019; Lee et al., 2022; Apr 2022 news). Variants that boost MSH3 expression pull onset forward, those that lower it push it back, while in HD mouse models, lowering this protein squelched repeat expansion, suggesting it might slow disease (Dragileva et al., 2009; Tomé et al., 2013; O’Reilly et al., 2023).     

For this study, co-first authors Emma Bunting and Jasmine Donaldson and colleagues put this MSH3-lowering strategy to the test in human neurons derived from a person with HD who carried 125 CAG repeats. Starting with iPSCs, they coaxed the cells to differentiate into medium spiny neurons, aka striatal projection neurons. They are among the first to degenerate in HD, and are a hotspot for somatic CAG expansion. The scientists treated these spiny-esque neurons with an MSH3-targeted ASO developed by Ionis Pharmaceuticals, Carlsbad, California. The drug dose-dependently lowered expression of the gene.

Would this curb somatic CAG expansion? To find out, the scientists exposed the cells to different doses of the MSH3-ASO, or a scrambled ASO, for 15 weeks, measuring the repeats’ length every three weeks. In controls, the number of repeats inched up from 125 to an average of 126.2 in that time. The MSH3-ASO reduced this expansion, with the highest dose halting it completely. The dose-response curve predicted that lowering MSH3 by 41 percent would be sufficient to halve CAG expansion, while an 83 percent lowering would stop it. Using CRISPR to knock out MSH3 also stopped the expansion and, in some cells, shrank it. Tabrizi told Alzforum that her lab is working to understand this shrinkage mechanism.

ASO Anyone? Striatal neurons, which express FOXP1 (yellow) and MAP2 (red), take up an MSH3 antisense oligonucleotide (green). Merge shown on left. [Courtesy of Bunting et al., Science Translational Medicine, 2025.]

Because the repeats grew only a bit over the 15-week period, the scientists put their ASO to the test in striatal neurons with a faster-growing repeat. To this end, they generated neurons lacking FAN1, a protein that protects against CAG repeat expansion (Goold et al., 2019; Goold et al., 2021). Knocking out FAN1 doubled the rate of CAG growth to about three extra repeats over 15 weeks. In this system, MSH3-ASO still kept it under wraps.

While the growth of CAG repeats in these cell-culture systems may seem minuscule, the process takes place over decades in the human brain, and may accelerate as the neuronal genome becomes less stable with age (Jul 2015 news; Oct 2018 news).

To test broader effects of knocking down MSH3, the scientists used bulk RNA-Seq. This turned up more than 800 differentially expressed genes in cells treated with MSH3-ASOs versus a nonspecific ASO. Two-thirds of these were downregulated, most being involved in cell development and axonal projection. Neither DNA damage response nor cancer pathways were affected, and the cells looked normal. Some of these genes were off-target victims of the MSH3-ASO, since other MSH3-ASOs did not provoke their downregulation, suggesting more specific ASOs might be found. Tabrizi told Alzforum she is optimizing MSH3-ASOs for use in people.

With an eye toward that, the scientists next generated human MSH3 knock-in mice, then injected MSH3-ASO or a nonspecific ASO in the lateral ventricles of their brains. Two weeks later, MSH3 expression had sunk across several regions. At the highest dose of 30μg ASO, MSH3 mRNA plummeted by 77 percent in the brain stem, 74 percent in the spinal cord, 49 percent in the cortex, and 46 percent in the striatum. This level of MSH3 lowering is predicted to substantially curb CAG repeat expansion, though this could not be directly tested in this mouse model.

The findings dovetail with another study, published February 8 in Cell. Its authors, led by William Yang, University of California, Los Angeles, knocked out several previously identified genetic modifiers of HD in a mouse model carrying 140 CAG repeats within the mouse Htt gene (Wang et al., 2025). Here, too, knockouts for MSH3 or the mismatch repair protein Pms1 most strongly affected HD phenotypes, including reduction of CAG expansion in striatal neurons, lowering of mouse Htt aggregation, and sparing striatal neurons.

Together with Tabrizi’s study, the findings converge on the idea that MSH3 and other DNA mismatch repair enzymes accelerate CAG expansion, and thus degeneration, in HD-vulnerable neurons.  

Both Tabrizi and Yang think that knocking down MSH3 could stave off HD pathology. Tabrizi noted that MSH3 also modifies other run-on repeats, including those causing fragile X syndrome, myotonic dystrophy, and spinocerebellar ataxias, and thinks this MSH3-targeted approach might work broadly for repeat-driven disorders, which number in the 60s.

“This work has important implications beyond Huntington’s disease,” agreed Michael Guo of the University of Pennsylvania in Philadelphia (comment below). His recent work tied short tandem repeat sequences to the polygenic risk burden of Alzheimer’s disease (Feb 2025 news).—Jessica Shugart

Comments

1. • Michael Guo
     Penn Medicine
   • Posted: 21 Feb 2025

   Over the last couple of decades, researchers have found that a tandem repeat that causes Huntington’s disease becomes unstable in certain vulnerable cell populations in patients’ brains, and that this instability drives disease progression. Moreover, we have found that genetic variation elsewhere in the genome modifies the tandem repeat instability, including at the MSH3 gene. Leveraging these genetic findings, the authors targeted MSH3 using antisense oligonucleotide therapy. They find that reduction of MSH3 levels ameliorates tandem repeat instability in both a cell-line model and a mouse model.

   The application of antisense oligonucleotide therapies is advantageous because this modality has already been used for established therapies for other neurological diseases. The authors’ work represents an important first step in showing that reducing MSH3 can reduce tandem repeat instability without causing obvious adverse effects on cell health. Future work will be needed to figure out if this therapy ultimately slows down progression of Huntington’s.

   This work represents an exciting translation of human genetic modifier findings to potential therapies for this devastating disease. It also has important implications beyond Huntington’s. There are approximately 60 other rare disorders, including forms of frontotemporal dementia, that are caused by a specific expansion in a tandem repeat. Many of these other disorders have also been demonstrated to display instability of the pathogenic tandem repeat in specific tissues. Thus, modifying levels of MSH3 and other DNA repair proteins represents a potential therapeutic strategy with applicability to many rare disorders beyond Huntington’s disease.

   Moreover, our team and others have recently identified that tandem repeats are expanded in Alzheimer’s disease. If instability of these tandem repeats also occurs in the brains of patients with Alzheimer’s, then therapeutic modification of MSH3 may have treatment implications for this very common condition.

   View all comments by Michael Guo

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References

News Citations

1. DNA Nuclease Fans the Flames of Huntington’s Disease 15 Apr 2022
2. Could Genetic Mosaicism in Adult Neurons Precipitate Disease? 15 Jul 2015
3. Islands of Mutated Neurons Dot the Brain. Are They Bad for Us? 23 Oct 2018
4. Expanded Repeat Sequences Raise a Person’s Risk for Alzheimer’s 12 Feb 2025

Paper Citations

1. Hong EP, MacDonald ME, Wheeler VC, Jones L, Holmans P, Orth M, Monckton DG, Long JD, Kwak S, Gusella JF, Lee JM. Huntington's Disease Pathogenesis: Two Sequential Components. J Huntingtons Dis. 2021;10(1):35-51. PubMed.
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4. Bates GP. Somatic CAG-repeat expansion drives neuronal loss in Huntington's disease. Neuron. 2025 Feb 5;113(3):342-344. PubMed.
5. Genetic Modifiers of Huntington’s Disease (GeM-HD) Consortium. Electronic address: gusella@helix.mgh.harvard.edu, Genetic Modifiers of Huntington’s Disease (GeM-HD) Consortium. CAG Repeat Not Polyglutamine Length Determines Timing of Huntington's Disease Onset. Cell. 2019 Aug 8;178(4):887-900.e14. PubMed.
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7. Dragileva E, Hendricks A, Teed A, Gillis T, Lopez ET, Friedberg EC, Kucherlapati R, Edelmann W, Lunetta KL, MacDonald ME, Wheeler VC. Intergenerational and striatal CAG repeat instability in Huntington's disease knock-in mice involve different DNA repair genes. Neurobiol Dis. 2009 Jan;33(1):37-47. Epub 2008 Sep 30 PubMed.
8. Tomé S, Manley K, Simard JP, Clark GW, Slean MM, Swami M, Shelbourne PF, Tillier ER, Monckton DG, Messer A, Pearson CE. MSH3 polymorphisms and protein levels affect CAG repeat instability in Huntington's disease mice. PLoS Genet. 2013;9(2):e1003280. PubMed.
9. O'Reilly D, Belgrad J, Ferguson C, Summers A, Sapp E, McHugh C, Mathews E, Boudi A, Buchwald J, Ly S, Moreno D, Furgal R, Luu E, Kennedy Z, Hariharan V, Monopoli K, Yang XW, Carroll J, DiFiglia M, Aronin N, Khvorova A. Di-valent siRNA-mediated silencing of MSH3 blocks somatic repeat expansion in mouse models of Huntington's disease. Mol Ther. 2023 Nov 1;31(11):3355-3356. Epub 2023 Sep 25 PubMed.
10. Goold R, Flower M, Moss DH, Medway C, Wood-Kaczmar A, Andre R, Farshim P, Bates GP, Holmans P, Jones L, Tabrizi SJ. FAN1 modifies Huntington's disease progression by stabilizing the expanded HTT CAG repeat. Hum Mol Genet. 2019 Feb 15;28(4):650-661. PubMed.
11. Goold R, Hamilton J, Menneteau T, Flower M, Bunting EL, Aldous SG, Porro A, Vicente JR, Allen ND, Wilkinson H, Bates GP, Sartori AA, Thalassinos K, Balmus G, Tabrizi SJ. FAN1 controls mismatch repair complex assembly via MLH1 retention to stabilize CAG repeat expansion in Huntington's disease. Cell Rep. 2021 Aug 31;36(9):109649. PubMed.
12. Wang N, Zhang S, Langfelder P, Ramanathan L, Gao F, Plascencia M, Vaca R, Gu X, Deng L, Dionisio LE, Vu H, Maciejewski E, Ernst J, Prasad BC, Vogt TF, Horvath S, Aaronson JS, Rosinski J, Yang XW. Distinct mismatch-repair complex genes set neuronal CAG-repeat expansion rate to drive selective pathogenesis in HD mice. Cell. 2025 Feb 8; Epub 2025 Feb 8 PubMed.

Further Reading

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