Developing an SCN8A ASO with PhD Student, Sophie Hill
An SCN8A Unraveled Story
Published by Sara Te, SCN8A Alliance Scientific Communications
This is a recap of our SCN8A Unraveled webinar with Sophie Hill. She is a 4th-year PhD student in Miriam Meisler’s lab at the University of Michigan.
The Central Dogma
Proteins do the actual work of a cell. For example, the sodium channel Nav1.6 is a protein.
The information for making proteins is in the DNA. When a genetic mutation occurs, it alters the DNA.
There is a step in between DNA and protein called mRNA, or a transcript. When a person has a mutation in the DNA, it is carried over by the mRNA and causes the cell to produce the wrong protein.
NaV 1.6 SCN8A Sodium Channel
The NaV 1.6 sodium channel is located in the cell membrane, the barrier of the cell. Their job is to allow sodium ions to pass through the membrane into the cell. Problems arise when the NaV 1.6 channel is mutated and allows sodium ions to pass through at the wrong time. For example, this gain of function mutation allows too many sodium ions in, which causes hyperexcitability, which is when neurons fire too frequently. This can cause seizures and result in developmental delays and other DEE symptoms.
What is an ASO?
ASO is short for antisense oligonucleotides, short stretches of DNA or RNA that can work in many different ways to reduce the amount of mRNA, which then leads to a reduction of its corresponding protein. There is an FDA-approved ASO for the treatment of spinal muscular atrophy called Spinraza. There is also an ASO currently in clinical trials for Dravet Syndrome, another DEE.
Sophie and the Meisler lab worked with Ionis Pharmaceuticals to design an ASO that reduces the abundance of SCN8A mRNA. The ASO works by binding to the SCN8A mRNA, telling another protein to break it down. This reduces the amount of Nav1.6 protein and hopefully makes up for the excessive sodium flow caused by the mutation.
How were the ASOs Tested?
Sophie and the Meisler lab injected the ASO directly into the brain of 2-day-old baby mice. Three weeks later, they harvested the brain and spinal cord of these mice and looked at the amount of SCN8A mRNA. Mice that received the control ASO (shown in black dots) had normal levels of SCN8A mRNA in the brain and the spinal cord. The control ASO is like a placebo, it’s not the real treatment. Mice that received the SCN8A ASO (shown in open dots) had reduced Scn8a mRNA levels, about 50% of control mice. So, the ASO did what they wanted!
Once they observed that the ASO reduced SCN8A mRNA levels, they wanted to test the ASO in a mouse that had an SCN8A mutation. They engineered these mice to carry the R1872W mutation, which was originally discovered in patients with developmental and epileptic encephalopathy (DEE). This graph shows the survival of a group of mice as a function of their age. On the x-axis (the bottom horizontal line) is the age of the mouse in days, and on the y-axis (the vertical line on the left) is the proportion of mice that are still alive.
The black line (on top) represents mice with the unmutated SCN8A gene, and they have normal lifespans, so none die before 42 days of age.
The blue line represents mice with the mutant SCN8A gene. All of these mice die around 2 weeks of age due to a single, lethal seizure. This mutation appears to be much more severe in the mouse than in humans. Because these mice die after their first seizure, they were unable to test how well the ASO prevents seizures.
Sophie and the Meisler lab took the same mutant mice and injected ASO into the 2-day-old baby mice. This first line shows mice that received a control ASO. Their survival is largely the same as untreated mice with the SCN8A mutation. All of the mice died around 2 weeks of age.
The second line shows mice that received our smallest dose of ASO, 15 ug. These mice lived about twice as long as control-treated mice!
The third line shows mice that received a middle dose of 30 ug. These mice lived for about 6 weeks.
The fourth line shows mice that received our largest single dose of 45 ug. These mice lived about 7 weeks, which is a 3.5x improvement in survival!
The final line shows mice that received 2 doses of ASO. These mice lived the longest of any group we tested, with average survival of around 9 weeks! We hope this means that we could keep giving doses and keep extending the lifespan. Although we were unable to test the effect of the ASO on seizures, we hope that the ASO would also reduce seizure severity or frequency.
Potential Side Effects
The problem is that SCN8A is a gene you can’t live without, so the level of its expression has to be continuously measured and adjusted. If the ASO dose is too high, this could cause movement problems.
Next Steps
- Find a pharmaceutical partner willing to produce and market the ASO
- FDA approval
- Clinical trial (Phase 1)
- Time Scale: YEARS
- Testing the ASO in other types of mouse epilepsy
- Treating mice with the ASO after they start having seizures
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