Fixing the Recipe: Lowering a Slice of Huntingtin

A new study shows that lowering a harmful piece of the huntingtin message (Htt1a) appears effective at reducing HD-related symptoms in mice, perhaps more so than targeting full length HTT. Whether lowering HTT1a in people has the same effect remains to be seen but studies in mice like this open up new ideas about HTT lowering. Let’s get in this study.

What are RNA transcripts?

Your body works by following a simple flow of genetic information. Back in 1957, Francis Crick described this as the fundamental process of biology: DNA replicates itself and serves as a template to create RNA, and RNA is then used to make proteins. RNA messages – known as transcripts – are molecules used as instructions to make proteins. Think of DNA as the written individual ingredients, RNA as the recipe, and proteins as the completed meal.

Scientists are especially interested in RNA when it comes to treating diseases with known genetic causes. Instead of trying to get rid of harmful proteins after they’ve already been made, it can be more effective to stop them from being made in the first place. This involves targeting the RNA molecules before they’re ever used to make proteins.

This idea is particularly important for Huntington’s disease (HD), as researchers are exploring ways to target the RNA that carries the instructions for making huntingtin (HTT), with the goal of producing less of the toxic protein and thus improving symptoms.

HTT1a: A toxic fragment

HD is caused by an expanded repeated section of the DNA letters C-A-G in the HTT gene. This expanded gene produces an abnormal version of the HTT protein, with an extra-long segment that causes it to fold incorrectly. Instead of doing its normal job, the misfolded protein becomes sticky and starts clumping together, damaging neurons and hastening symptoms. Think of it like following a key lime pie recipe where one instruction is accidentally repeated over and over: adding one teaspoon of salt makes it tasty, but an extra twenty will ruin the pie.

But that’s not the whole story. The genetic mutation doesn’t just affect the final protein – it can also interfere with how the gene’s instructions (messenger RNA, or mRNA) are made. Sometimes, the instructions are cut off early, which produces a shorter version of the HTT message called HTT1a. This creates a tiny and potentially harmful version of the huntingtin protein. This shorter HTT1a protein clumps together even more easily and potentially causing more toxicity in brain cells. Imagine that alongside adding salt repeatedly, your recipe includes whole limes (peel and all) along with lime juice. At that point, the entire pie is inedible!

All of this might sound like a recipe for disaster, but it also points to a possible solution. Instead of trying to fix the final, damaged product, researchers are going straight to the source: mRNA, the recipe itself.

siRNA: Biology’s eraser

siRNA, or small-interfering RNA, is a tool that scientists use to reduce the amount of mRNA in a cell. By targeting and breaking down specific mRNA, siRNA effectively prevents certain proteins from being produced. If mRNA is the recipe for a dish, siRNA acts like a pencil that erases some ingredient words so that you just have to skip them. Without all the extra tablespoons of salt and the whole limes, your pie will turn out as planned (hopefully delicious).

It is still unclear whether targeting full-length HTT, HTT1a, or both might improve HD outcomes in people. There are currently multiple drugs in development that use these different methods: AMT-130 (uniQure), ALN-HTT2 (Alnylam), and V0659 (Vico) target both full-length HTT and HTT1a, whereas tominersen (Roche), votoplam (Novartis), and SKY-0515 (Skyhawk) likely target only full-length HTT. Tominersen is the only HTT-lowering therapeutic that has been evaluated in an advanced (Phase 3) clinical trial which is looking for how the drug might alter symptoms in hundreds of people. Although it did not show any benefit in the complete cohort dataset, there were signs of improvement in some participants, so the community is waiting for the results of the next Phase 3 trial currently underway for this drug. 

What was investigated?

A study published last month explores whether siRNAs that target full-length HTT or HTT1a transcripts can improve signs of HD in mouse models, and whether targeting one, the other, or both works best. In an earlier study by the same group of researchers, they reduced levels of these genetic messages by genetically altering the mice. However, using an injectable drug has greater potential for how this might pan out in real-world use in people.

The researchers designed two siRNAs – one that targets full-length HTT, and one targeting HTT1a. After confirming that these siRNAs work, they injected them into the brains of mice that model HD. In HD research, different types of HD mice are used depending on the features that they have. The one they used in this study has some features that mimic those people of HD that progress over time, like the appearance of HTT aggregates, especially in the nucleus of brain cells, and dysfunction in how networks of genes turn on and off. To evaluate how effective this approach was, researchers designed an interventional study and organized the mice into three main groups:

• Early group: injected in the very earliest stages of disease (2 months old) and analyzed as signs and symptoms were worsening (6 months)
• Double group: injected in the early and middle stages of disease (2 and 6 months) and analyzed at the peak of symptoms (10 months)
• Late group: injected in the middle stage of disease (6 months) and analyzed at the peak of symptoms (10 months)

These groups were compared to see whether earlier treatment, later treatment, or repeated dosing led to better outcomes.

Highlighting the hippocampus

After injecting each mouse cohort with the siRNAs designed to target full length Htt or Htt1a, they waited several months and measured the amount of HTT and HTT1a protein in different parts of the brain. Compared to untreated mice, they found that both were most effective in the hippocampus, the part of the brain that is responsible for emotion and memory. 

This is not typically where HD pathology is studied; research usually focuses on the striatum, which primarily controls motor and executive functioning. Although the researchers did examine the striatum, they observed only modest reductions in HTT and no change in HTT1a levels. But HD affects the whole brain, and scientists often follow the data where it takes them! Because their findings led them to the hippocampus, it became the focus for the remainder of the study. 

While both siRNAs reduced the protein in the hippocampus, targeting Htt1a seemed to have some additional benefit for one sign of HD: it delayed HTT protein clumping. There were fewer aggregates formed, and for mice in the Double treatment group, aggregates in the nucleus did not appear at all. Overall, only the HTT1a treatment seemed to reduce both the amount and severity of the toxic protein accumulations in the hippocampus. 

That said, both treatments had a positive effect on HD-related gene activity in mice within the Early and Double cohorts, though the Htt1a siRNA seemed to have a more pronounced effect. However, the Late group showed little benefit of treatment and some signs of disease may even have worsened. Overall, the results suggest that this approach works best when given early, and appears to be less effective if started later in disease progression.

Why does this matter?

This study shows that the way full-length HTT and HTT1a proteins are involved in forming HTT aggregates is complex and can differ depending on the type of brain cell involved (hippocampus versus striatum). However, most of the benefits seen in the experiments they conducted come from lowering HTT1a, which should be a consideration when designing HD therapeutics. Overall, this suggests that future treatments may work best if they reduce both HTT and HTT1a, or if they are designed to specifically target HTT1a in humans.

Some caveats

As promising as these findings are, this study was conducted in mice, not people. HD mouse models develop disease over months rather than decades and often carry very large CAG expansions in every cell, which may lead them to produce more HTT1a than is typically seen in people with HD. This means HTT1a could play a larger role in these mice than it does in human disease. The strongest benefits were also seen in the hippocampus rather than the striatum, a brain region more closely linked to many HD symptoms. Future clinical studies will be needed to determine whether lowering HTT1a has similar benefits in people.

Summary

• In HD, faulty RNA can produce both the full huntingtin protein and a shorter, potentially more toxic version called HTT1a, which might contribute to HTT toxicity and symptom onset.
• In this study, researchers developed siRNA treatments that lowered HTT and HTT1a levels in mouse brains, reduced toxic protein clumping, and improved abnormal HD gene activity.
• Targeting HTT1a seemed to be more effective than targeting full-length HTT in mice that model HD by some measures, although lowering HTT1a and full-length HTT produced benefit by looking at other signs of HD in these models. 
• Overall, the results suggest that reducing HTT1a could be important, but the jury is out on which approach might be better in people where the amount of HTT1a made is very different.