Turning down mismatch repair genes slows Huntington’s repeat growth in human neurons

Scientists used cells from a person with Huntington’s disease (HD) to test whether lowering the levels of DNA repair genes can slow the growth of the nefarious CAG repeat. In these human cells grown in the lab, the repeat did grow more slowly, suggesting a potential therapeutic strategy to possibly delay the onset and progression of the disease, although this work is still at an early stage.

Why expanding CAG repeats matter in HD

HD is caused by an abnormally long stretch of three DNA letters, CAG, in the huntingtin gene, often shortened to HTT. Generally, the increasing length of the expanded CAG repeat is associated with people showing symptoms of HD earlier and faster disease progression. 

We now know that for people with HD, the CAG length a person inherited is not fixed for life. In some cells, especially in a brain region about in the middle of the head, called the striatum, the CAG repeat can slowly grow longer over many years. This process is called somatic expansion – expansion of the CAG repeat in “somatic” cells, or cells of the body.

Evidence from human brain tissue and animals that model HD suggests that this extra growth (somatic expansion) is a significant driver of vulnerability of these neurons and is a major factor in driving disease progression.

DNA repair: a key driver of CAG expansion

Our DNA is constantly being damaged by things like UV exposure from the sun, environmental pollutants, and stress inside our cells. But cells are always working to repair this damage in their DNA. One critical system responsible for maintaining the integrity of the genetic material is called Mismatch Repair (MMR). It acts like a molecular proofreader, locating and correcting small errors to maintain genetic integrity.

However, the highly repetitive CAG sequence in the HTT gene is structurally unstable and can form unusual shapes, such as loops of DNA. When components of the MMR system encounter these odd structures, the repair process is thought to misread the DNA letter code, kind of like the proofreading system accepting typos. 

These proofreading errors by the MMR system can lead to the incorporation of additional repeats. In this context, the MMR system, which normally protects the genetic code, inadvertently becomes a major mechanism driving the harmful expansion of the CAG repeat.

This destructive role of MMR in contributing to HD is strongly supported by long-standing research. Large-scale genetic studies, known as Genome-Wide Association Studies (GWAS), in people with HD confirmed this link by identifying natural genetic differences (variants) in several MMR-associated genes that modify the age at which symptoms begin. 

These findings established compelling therapeutic strategies. If we can safely reduce the activity or levels of specific MMR genes, we might be able to effectively slow this expansion process at its source.

Studying mismatch repair in human HD Neurons

Most earlier research on mismatch repair and CAG expansion used mice that model HD or animal cells grown in a dish. In a recent study led by Dr. Sarah Tabrizi from University College London, the researchers used a system more directly relevant to humans. 

They began with skin cells donated by a seven-year-old girl with juvenile onset HD, who carried a very large expanded CAG repeat of over 125 in the HTT gene. The scientists added molecules that coaxed the skin cells into becoming stem cells, which are capable of self-renewal and can be directed to become various cell types, including brain cells.

Using protocols that add different chemicals at different points in time, the team converted the HD stem cells into brain cells enriched for medium spiny neurons (MSNs) that are found in the striatum, a central brain region heavily affected by HD. MSNs are specific brain cells that are known to be particularly vulnerable to degeneration in HD. The researchers consistently measured the CAG repeat length in both the dividing stem cells and these brain cell cultures to monitor how the repeat length changed over time.

Using a molecular dimmer switch

To test the role of mismatch repair, they used a powerful genetic tool called CRISPR interference (CRISPRi). CRISPRi acts as a molecular system for precisely reducing gene activity in single genes at a time. This system is distinct from standard gene editing because it does not cut DNA. Instead, it uses a molecule that you can think of as molecular scissors, called Cas9, that is stapled at the molecular level to a dimmer switch that effectively lowers levels of a target protein. 

The team used CRISPRi to reduce the activity of mismatch repair genes that were identified in the human genetic studies (GWAS). They also targeted the partners of those MMR genes. The targeted genes included components from the multi-protein complex families, the MutS family (MSH2, MSH3, MSH6) and the MutL family (MLH1, PMS1, PMS2, MLH3), along with the enzyme, DNA ligase 1 (LIG1). 

The goal of the study was to reduce their activity to levels feasibly achievable by therapeutic drugs, rather than eliminating the genes completely. This approach allowed them to more realistically mimic what could be developed as a treatment or imitate those variants found to be naturally occurring in people with HD who had later onset of symptoms.

Next the researchers checked to see that their experiments were successful, so they did another experiment called an “MMR deficiency assay.” This allowed them to confirm that they were actually lowering activity of the MMR target genes. To do this, they treated the cells with a chemical that mimics DNA damage. In cells that have a fully functioning MMR system, this chemical typically triggers cell death. But if MMR function is compromised, cells are able to survive. 

The experiment confirmed that the MMR system in the modified cells was weakened, showing increased cell survival compared to controls. Importantly, the reduced function was not total, aligning with the goal of partial reduction and giving the researchers a system where they can molecularly dim the levels of these genes to turn them down rather than off.

The impact of dimming MMR genes on CAG expansions

Finally, they looked at the rate at which the CAG repeat expanded in both the dividing stem cells and the specialized striatal neurons, comparing the expansion rates with and without the reduced activity of the mismatch repair genes.

In the HD stem cells, the CAG repeat normally grew longer over time, which was expected. But when several mismatch repair genes were turned down with CRISPRi, this growth slowed, suggesting the team’s theory was correct! The strongest slowing effects were seen when the team reduced the major MutS components (MSH2 and MSH3) and the core MutL component (MLH1). Lowering these genes reduced the rate of expansion in the dividing stem cells by between 60% and 65%, with other MutL factors (PMS1, PMS2, and MLH3) reducing the rate by 25-35%.

Most importantly, similar patterns were seen in the brain cell cultures made from the same HD stem cells. The researchers strategically focused on the MutL factors (PMS1, PMS2, and MLH3) in neurons, as earlier stem cell experiments ruled out MSH6 and LIG1, and MSH2 posed potential safety concerns due to cancer risk. They also didn’t do a deep dive on MSH3 since it’s being actively pursued as a therapeutic target elsewhere. 

In the neurons, with MLH1 turned down, this expansion slowed by 69%, and targeting PMS1, PMS2, or MLH3 also slowed the expansion by over 20% compared with control cells. This shows that mismatch repair remains a key driver of somatic expansion in human HD neurons, not only in dividing stem cells or in mice. Additionally, it shows that by adjusting levels of these specific MMR genes, researchers may be able to meaningfully slow somatic expansion. A very exciting prospect!

What the study found and what it does not show

In plain terms, the core message of this research is: In human neurons grown in a dish that were created from someone with HD, turning down mismatch repair genes seems to be enough to slow the growth of the harmful CAG repeat.

This work is encouraging for the idea of “anti-expansion” therapies. At the same time, it is important to be clear about what this study does not show. All the work was done in cells in dishes, not in living brains. Only one juvenile onset HD stem cell line, with a very long repeat, was used. 

The study measured CAG repeat length and its distribution. It did not test neuron survival, behavior, or brain function. It also did not address long-term safety. Mismatch repair genes help protect all tissues from cancer, and changing their activity in people carries risks of causing cancer, especially since the loss of genes like MSH2 and MLH1 is associated with a significantly increased risk of nervous system cancers, with PMS2 also carrying some risk.

What the work does provide is a more realistic shortlist of mismatch repair genes that look promising in a human HD context, highlighting members of the MutL family, particularly PMS1, as potential targets. It supports the idea that carefully reducing the activity of selected mismatch repair genes, probably to a moderate degree and possibly in combination, might be a valuable way to slow CAG expansion. This information could help guide the design and prioritization of future therapies that aim not just to treat symptoms, but to change the pace of Huntington’s disease itself.

Summary

• Mismatch repair drives CAG expansion in human neurons: Using brain cells created from a person with HD that were grown in a lab dish, researchers confirmed that the mismatch repair system drives CAG repeat growth in human neurons, not just in mice or dividing cells.
• Turning down repair genes slowed expansion: By partially reducing the activity of specific mismatch repair genes, the team slowed CAG repeat growth by up to 69% in neurons, with members of the MutL family, particularly PMS1, emerging as potential therapeutic targets.
• Genetic findings now have experimental support in human cells: Large genetic studies previously pointed to mismatch repair genes as modifiers of HD onset. This study confirms their role in human neurons grown in a lab dish and helps prioritize which genes to target. The work was done in a single cell line in a dish, but it is an important step toward therapies that aim to slow CAG expansion.