Break Up With Your CAGs: How Three Letters Could Change Huntington’s Disease

Huntington’s disease (HD) is caused by a repeated stretch of the genetic letters C-A-G within the huntingtin (HTT) gene above a critical number. If the repeats exceed 40, then signs and symptoms of HD will begin at some point in that person’s life, if they live long enough. The disease-causing CAG stretch expands throughout life, particularly in vulnerable brain cells, which scientists think eventually triggers cell death. 

New research used cutting-edge gene editing to create human stem cells with different CAG repeat lengths and genetic spellings. They then tracked how these repeats changed over time using advanced sequencing technology. The team discovered that inserting multiple genetic “interruptions” into the CAG repeat, breaking up the pure stretch of CAGs, had major benefits. What exactly did they find and what does this mean for future therapeutics? Let’s find out!

A Cellular Time Machine

Imagine watching a disease unfold in slow motion so you could track the exact moment when things start to go wrong. That’s essentially what researchers at the University of Milan have created – a platform to watch HD develop at the cellular level, repeat by repeat, day by day.

The team, led by Dr. Elena Cattaneo, engineered human stem cells carrying different versions of the HTT gene. Using CRISPR gene editing, they swapped in HTT sequences with various CAG repeat lengths, ranging from 21 repeats (below the disease threshold) up to 107 repeats (well into the disease range).

They called this collection of cell lines the “CAGinSTEM platform,” and it could become a powerful tool for understanding how CAG repeats behave over time.

Watching Repeats Grow

One of the trickiest aspects of studying CAG repeat instability has been measuring the expansion accurately. Traditional sequencing methods can struggle with repetitive DNA. Imagine trying to accurately count 42 of the same letters in a row. It’s likely at some point you may question if you were on 31 or 32 and have to start over. The same process happens in an experiment when molecular machines try to read the number of CAG repeats.

The researchers solved this problem using a specialized type of sequencing that can read very long stretches of DNA in a single pass, maintaining information about the exact sequence composition.

Over 120 days of growing cells in dishes, the team observed that cells that start with 81 and 107 CAG repeats showed steady, linear expansion of their repeats. In contrast, cells with 45 or fewer repeats remained stable, with no major changes to their CAG number. When they turned these stem cells into striatal neurons, the brain cells most affected in HD, they saw similar patterns, with the 107 CAG line showing expansion even in neurons.

Looking at cells before and after they became neurons allowed the researchers to determine if cell division was influencing CAG expansion. While stem cells divide again and again to create more cells, most neurons don’t – they’re what scientists called “post-mitotic,” meaning “after mitosis” or “after cell division”. Because CAG expansion remained at very high repeat numbers both before and after the cells became neurons, it suggests cell division isn’t the contributing factor.

The Power of Interruption

Here’s where the study gets really interesting. Most people (over 95%) have a natural interruption in their CAG repeat: it reads CAG over and over until the end of the repetitive section, where it reads CAG-CAA-CAG, with that single CAA near the end. Previous studies in people have shown that losing this CAA interruption leads to earlier disease onset, while having an extra CAA delays onset. 

The researchers tested this directly in their cell platform. They created lines with 107 pure CAGs (no interruption), lines with the typical single interruption, lines with 2 CAA interruptions, and (most dramatically) lines with 4 CAA interruptions strategically placed throughout the repeat.

The results were striking. The double CAA interruption reduced instability compared to the standard single interruption. But the 4 internal CAA interruptions appeared to completely abolish repeat expansion over 120 days. The repeats simply stopped growing, both in dividing cells and in neurons. Quite intriguing!

More Than Just Stability

Stopping repeat expansion would be valuable on its own, but the researchers also discovered that the multiple CAA interruptions had other benefits, as they appeared to prevent several HD-related problems in the cells.

Neurons with the 107 CAG repeat with the regular 1-CAA interruption showed difficulty developing into the right type of neuron. They had fewer markers defining them as striatal neurons and more markers from a different brain region, suggesting their development into this specific type of neuron was a bit confused. These findings are in line with work from other labs using brain samples from people, which have shown an erosion of cellular identity of this type of neuron with expanding CAG repeats.

However, the 4-CAA-interrupted line seemed to maintain normal striatal neuron development. This suggests that 4 CAA interruptions preserve the genetic identity of the striatal neurons!

The team also examined how the DNA and other molecules were organised in a region called the nucleus of the cells, an area of growing interest in HD research. Cells with 1 CAA interruption in 107 repeats had a smaller nucleus on average, more compact DNA that isn’t turned into protein, and disrupted structures important for regulating which genes stay turned off during development. The 4-CAA interruptions normalized all of these features, restoring nuclear size, DNA organization, and features used to control levels of different genes.

Interestingly, some cellular disease aspects weren’t improved by the CAA interruptions. Neurons with interrupted repeats still showed abnormal cell shape similar to the cell line with 1 CAA interruption in 107 repeats, with shorter neuron branches (dendrites) and smaller cell bodies. This suggests that these particular features may depend on the protein encoded by the HTT gene and its repeats, rather than on DNA instability or repeat purity.

The DNA Matters, Not Just the Protein

For many years, HD research focused almost exclusively on the toxic protein. But this study reinforces a paradigm shift happening in the field: the DNA sequence itself, including its purity and tendency to expand, seems to also play a direct role in disease.

And here’s where it gets a little bit wild – CAA and CAG both code for the protein building block glutamine. So inserting CAA interruptions doesn’t actually change the protein! Yet these interruptions seem to prevent repeat expansion and prevent cellular problems. We told you it was wild…

This seems to support the “two-stage” model of HD as it relates to the CAG expansion: you inherit a CAG repeat that isn’t overtly toxic initially, typically allowing for decades of healthy life, but it expands over your lifetime in certain brain cells until it crosses a threshold and triggers cell death.

While some researchers have theories about what exact length triggers toxicity related to CAG expansion and how exactly this happens, no one knows for sure. One theory is that the pure CAG repeat forms stable DNA structures that promote slippage and expansion when the gene is copied. CAA interruptions could disrupt these structures, preventing the expansion process.

A Therapeutic Possibility?

The findings from this recent work raise an intriguing question: could introducing CAA interruptions be therapeutic? Recent proof-of-principle studies have used CRISPR base editing to convert some CAGs to CAAs in cells and mice, with encouraging results. However, translating gene editing to post-mitotic human neurons in living brains faces enormous technical challenges – delivery efficiency, precision, and safety all remain major hurdles.

Perhaps more immediately, the CAGinSTEM platform itself offers value for drug discovery. Researchers can now screen for potential medicines that either reduce repeat instability or mitigate its downstream cellular effects, using these well-characterized, quality-controlled cell lines that seem to faithfully recapitulate some aspects of HD pathology.

Natural Protection?

The study also hints at an intriguing possibility that some people might carry naturally occurring internal CAA interruptions that protect them from disease despite having pathogenic-range CAG repeats.

While never observed in the existing databases with information on people who have HD, such protective variants could exist in presymptomatic individuals who never develop symptoms.

The Bottom Line

It’s important to note that studies like this, that grow a specific type of cell alonel in a dish, don’t recapitulate what’s happening inside the brain, which is made up of many different cell types all connecting and communicating with each other. These types of studies are good at getting an idea of what certain types of cells are doing on their own, and how those disease-related changes could contribute to and impact the entire system.

This study adds evidence to other work that suggests CAG repeat purity directly affects both repeat instability and cellular dysfunction in HD, while developing a tool researchers can use to ask questions around this finding.

By preventing the formation of long, pure CAG stretches through strategic interruptions, researchers may be able to block repeat expansion and prevent multiple HD-related effects in neurons, all without actually changing the glutamine protein length. Wild!

The work continues to shift our understanding of what drives HD pathology, emphasizing that it’s not just about the protein you make, but about the DNA sequence you inherit and how it changes over time. While therapeutic applications for these findings remain speculative, the CAGinSTEM platform offers researchers a powerful new tool for understanding HD mechanisms and testing potential interventions.

Summary

• The platform: Researchers created quality-controlled human stem cell lines with different CAG repeat lengths and compositions in the huntingtin (HTT) gene
• Advanced tracking: Using long-read DNA sequencing, they measured CAG repeat changes over time in both dividing cells and neurons
• Length matters: Cell lines with 81-107 CAG repeats showed linear expansion over time, while shorter repeats remained stable
• Pure vs. interrupted: Standard repeats with one CAA interruption near the end still expanded; adding a second CAA interruption reduced expansion
• Complete blockade: Inserting 4 CAA interruptions throughout the repeat seemed to stopp expansion in both dividing cells and post-mitotic neurons
• Cellular rescue: The 4-CAA interruptions prevented multiple HD cellular effects, including impaired striatal neuron development, disrupted nuclear organization, and altered gene levels, all without changing the glutamine protein length
• DNA-driven disease: The findings contribute to the theory that repeat purity and instability, not just polyglutamine protein length, directly drive HD pathology
• A research tool: The CAGinSTEM platform offers a robust system for studying HD mechanisms and screening potential therapies