A road less traveled: how making less huntingtin can alter somatic instability and may delay symptoms

One mystery that many scientists think holds the key to curing HD is its mysterious age of onset. Although people with HD carry the expanded gene from birth, they generally don’t develop symptoms until later in life, suggesting something bad is brewing beneath the surface! One explanation, which has gained significant traction in recent years, is a process called somatic instability, where the expansion worsens over a person’s life. Recent work from the lab of Dr. Jeff Carroll at the University of Washington investigated several genetic techniques to understand what causes somatic instability and whether huntingtin-lowering therapeutics might slow it down. 

An Unstable Repeat

To understand somatic instability, let’s briefly revisit how genes work. Normally, genes like huntingtin, or HTT, are copied to make messenger molecules, called mRNA, through a process known as transcription. These genetic messages can then be used as a template to make proteins through another process called translation. 

However, in HD, the HTT gene contains extra genetic letters (C-A-Gs) that repeat too many times, causing its mRNA message to create an abnormal protein. In some cells, these repeating CAGs can grow even longer over someone’s life, leading to mRNA that is increasingly repetitive. By the time symptoms appear, these CAG repeats may have grown into the hundreds in certain cells. The continuously expanding CAG repeat in HTT, called somatic instability, is a leading theory for why the onset of HD is typically delayed into adulthood. 

Many ongoing clinical trials are focused on reducing the amount of HTT produced from the faulty gene. However, it’s unclear if lowering HTT levels will slow down the growth of the CAG repeat in the HTT gene. Although somatic instability is a prime suspect for causing HD’s delayed onset, it’s still only a correlation. Regardless, it’s certainly worth investigating what causes it and whether HTT-lowering therapies, which are already in clinical trials, can affect it. 

Dialling Down Huntingtin 

In a new study, a team at the University of Washington tested whether HTT lowering affects somatic instability. From previous work, they had used a type of therapy called Antisense Oligonucleotides (ASOs), which bind mRNA and send it to the cell’s trash can, to lower HTT levels in mice. They followed up on these experiments and discovered that ASOs also reduced CAG repeat growth by about 50%. This is good news because several ongoing clinical trials are already investigating ASOs.

Although the ability of ASOs to reduce target mRNA levels is well understood, the researchers were surprised that it stunted the growth of CAGs in the HTT gene. They suspected the ASOs might also disrupt mRNA at its source – a process called transcription. Recent work by other groups has linked rates of transcription with the growth of CAGs, such that the more the HTT gene is used to make mRNA, the quicker the CAGs build up. This hypothesis led the team to investigate exactly how ASOs were slowing CAG growth. 

The researchers considered two possible ways ASOs might be slowing somatic instability. 

1. The HTT protein itself was responsible for somatic instability, and by reducing the production of HTT, ASOs reduced somatic instability. 
2. The process of switching on the HTT gene was causing somatic instability, and by reducing transcription, ASOs reduced somatic instability. 

To find out how ASOs might affect somatic instability, the researchers injected a similar molecule into mice, called siRNA, which reduces HTT protein but does not affect transcription. When HTT protein levels were lowered using siRNA, they did not see any effect on somatic instability. This doesn’t mean siRNA wasn’t exerting a beneficial effect, just that siRNA wasn’t reducing somatic instability in the cells the team looked at. However, it does indicate that ASOs are slowing CAG growth by disrupting transcription, and not by lowering protein levels. 

Fewer Deliveries, Less Potholes?

To visualize the difference between siRNA and ASOs, imagine the HTT gene as an old road traveled by semi-trucks making deliveries, and the packages represent mRNA messages. With each year that the road is driven on, its potholes and cracks worsen, just as HTT’s CAG repeat worsens the more it’s used to make protein. Reducing HTT levels with siRNA is like reducing the number of packages, but the same number of trucks are still on the road – they are just emptier! ASOs, however, reduce the number of trucks, and fewer trucks mean less wear and tear on the road, and thus slower CAG growth. 

The researchers tried a more direct approach to test the connection between somatic instability and transcription. They turned to a genetically modified mouse model of HD where HTT transcription can be switched on or off, like a switch, by adding a special chemical to their drinking water. In mice where HTT transcription was switched off, they observed somatic instability slowing down. In addition, the longer HTT transcription was turned off, the less the CAG repeats grew. These results, in addition to their ASO experiments, provided good evidence that transcription was partially responsible for somatic instability. 

Zinc Finger Roadblocks

Although switching HTT on or off by adding a chemical to drinking water sounds fantastic, it only works in this specific type of genetically modified mice, which we sadly are not! So the researchers turned to a more practical approach using Zinc Finger Proteins (ZFPs), which are genetically modified proteins that attach directly onto CAG repeats and block transcription. From our analogy, ZFPs are like giant roadblocks cutting off traffic. If the delivery trucks driving over the road (representing transcription) are causing the potholes to worsen (CAG growth), then halting the traffic should slow somatic instability. 

To test ZFPs, they used a virus to deliver their DNA instructions into mouse brains. One side of the mouse’s brain got a version of the ZFP that latches onto the CAG repeat and shuts down transcription, and the other side got a version of the ZFP that binds HTT but does not shut down transcription. The ZFPs that block transcription showed an impressive 70% reduction in somatic instability. Surprisingly, ZFPs that bind to HTT but don’t block transcription still had a modest 42% reduction in somatic instability. This is good news because completely shutting down HTT transcription might be unsafe because HTT still performs important functions inside brain cells. So keeping HTT partially on while slowing somatic instability might represent a safer therapeutic approach. 

Therapeutic Directions

Collectively, these results show that dialing down HTT’s transcription not only reduces the amount of toxic HTT protein in the cell but might also slow its CAG growth. Although slowing CAG growth sounds like a home run, it’s important to reiterate that we still don’t know for sure if somatic instability is causing disease onset – it’s just a promising lead! In addition, reducing HTT transcription, which was linked to slowed somatic instability, might cause entirely unrelated problems in the cell. In our analogy, blocking package deliveries would stop the potholes from forming, but this would also surely create an angry bunch of customers waiting for their packages! 

Clinical trials using ASOs are already underway, and therapies based on ZFP are being worked on. Although there’s plenty of room for optimism, there are some important caveats. First of all, the mice used in these experiments are genetically engineered with an extreme CAG repeat mutation, because they otherwise wouldn’t show symptoms due to their short lifespan. And whether these therapies will translate effectively or safely into humans is another big question mark. For example, although ASOs and ZFPs might be tolerated within the very short lifespan of a mouse, we don’t know the long-term safety or effectiveness in humans. Regardless, we’ll be following every development closely and sharing updates as soon as they are released! 

Summary

• CAG repeats in the HTT gene keep expanding over life, and this somatic instability may contribute to HD’s delayed onset.
• ASO treatments slow repeat expansion by reducing HTT transcription, not just HTT protein levels.
• Multiple experiments, including siRNA, switchable transcription, and Zinc Finger Proteins, confirm that less HTT transcription means less CAG growth.
• Therapies targeting transcription look promising, but it’s still unclear whether slowing somatic instability will change HD onset in humans.

Learn more

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