Building An ASO For TUBB4A-Related Leukodystrophy (Pt. 1)

A conversation with Dan Williams, Ph.D., CEO, SynaptixBio

Developing a therapy for an ultra-rare neurodegenerative disease requires more than identifying a genetic mutation. It requires a deep understanding of disease biology, a therapeutic strategy capable of modifying that biology, and a development pathway realistic enough to reach patients.

For SynaptixBio, that journey began with TUBB4A leukodystrophy, a devastating pediatric disorder that currently has no approved treatments. The company's approach centers on antisense oligonucleotides (ASOs) designed to silence the expression of the disease-causing mutated TUBB4A gene.

In Part 1 of Life Science Connect Acquisition Editor Michael Soloway’s conversation with SynaptixBio CEO Dan Williams, they discuss the biological rationale behind the program, how the company selected its lead ASO candidate, and the translational evidence supporting advancement toward the clinic.

ARNA: SynaptixBio is focused on gene silencing via antisense oligonucleotides. What made ASOs the right modality for TUBB4A leukodystrophy?

DW: Let me first talk about the disease itself. TUBB4A leukodystrophy is a debilitating, life-limiting neurodegenerative disease that mainly affects babies and young children. People with TUBB4A-related leukodystrophy show progressive deterioration of motor skills, including walking, sitting and using their hands, but it also extends to speech and swallowing. Symptoms and progress differ depending on when the disease first appears.

When symptoms begin in the first few months of life, children mostly never reach early motor milestones like head control, sitting, or walking. The effect of the disease also tends to be severe and its progress more rapid.

When symptoms begin later, in early childhood, children start losing their previously achieved milestones and the progression may be slower. In these cases, the primary symptom is progressively greater trouble with movement.

There is nothing available at the moment that treats the disease, though physiotherapy can make life easier, but there is nothing that targets the actual disease itself. That’s the unmet need we’re driven to find. And, as it happens, one of my friends and colleagues, one of the company’s cofounders, has a daughter with the disease.

We chose ASOs because we believe gene silencing is the best way of tackling a single gene mutation like TUBB4A leukodystrophy; the vast majority of rare diseases are monogenic.

Gene editing would be another way of targeting the disease, but this potentially has more side effects. In addition, we wanted to develop something that we could bring to market as quickly as possible, and ASOs are proven; they’re known. They are safe and have a very high probability of success, something we have seen in trials with mice and non-human primates. As a young company, we wanted something that would get ourselves toward the clinic to do what we can for these children as early as we possibly could.

ARNA: How did the scientific understanding of the disease shape your therapeutic hypothesis?

DW: The disease is relatively young from the point of view of discovery. Professor Adeline Vanderver at the Children’s Hospital of Philadelphia (CHOP) identified the mutations around the disease. In 2015, she started making breakthroughs with TUBB4A leukodystrophy. In 2021, the first thing we did was initiate a collaboration with Professor Vanderver to produce more data and push that toward commercialization. She did some very elegant experiments, looking at knocking out the gene using CRISPR. What she found was that myelination was occurring with the wild type but wasn’t occurring with the mutation. This led to the hypothesis that gene silencing would be the way to move forward.

From there, it was a case of looking for molecules that could affect the silencing. That’s where ASOs came in. You can use them to knock down the mRNA that’s produced from the mutated TUBB4A gene, which in turn stops the production of toxic proteins that result in insufficient myelin to surround the nerve cells in the brain.

The other part of the hypothesis is that tubulins or other tubulin genes come in and replace it. This way, we get less expression of the aberrant protein and more expression of the tubulins that step into its place and enable myelination. At this point, they tend to be quite functional when you get them into the clinic and into patients.

ARNA: What were the most important scientific questions you needed to answer before selecting a lead ASO?

DW: As I’ve mentioned, the design of the ASO must enable it to knock down the mRNA within the cells. The first thing we screened for was which ASO caused a greater than 50% decrease of the mutated TUBB4A gene expression within cell lines in vitro. Then we wanted to make sure that efficacy was seen in vivo. Again, that translation is extremely important because we’re also looking at biodistribution and tolerability. The therapeutic is going into children, so everything we think about every step of the way is how safe any treatment is going to be for them.

We also thought about the best way to dose this and looked at various in vitro safety markers. Some ASOs go straight to the liver and cause hepatotoxicity.

Neurotoxicity is a critical consideration because that’s where we’re targeting. Another thing that we factored in was the potential to cross-react, because if you’re designing a piece of DNA/RNA that is complementary to a sequence within a gene that you’re looking for, it doesn’t mean there aren’t similar sequences elsewhere in other genes. We didn’t want the molecule binding to those genes, knocking them out in the process, and causing other problems. As a result, we did a lot of in silico cross-reactivity work to figure out if there were other genes that would be hit by the ASO, how important those genes are, and what would happen if we knocked them down. This was all factored into the big picture of which molecules to take forward.

ARNA: How did you balance potency, specificity, and safety during optimization?

DW: One of the biggest lessons we learned during optimization was that the most potent molecule isn’t always the best therapeutic candidate. Early on, we identified several ASOs that looked exceptionally promising in vitro. At first, we thought, these are exactly what we’re looking for. But as we progressed into more advanced testing, some of those highly potent molecules began to show signs of toxicity, particularly at higher doses.

That reinforced the importance of balancing efficacy with safety. Ultimately, we selected a molecule that provided robust target knockdown while maintaining a favorable safety profile, rather than simply choosing the most potent candidate.

Specificity was equally important. We used in silico analyses to identify potential off-target binding across the genome and eliminated several candidates because they showed possible cross-reactivity with genes whose biological functions were poorly understood. Even if we couldn’t definitively say those interactions would be harmful, we weren't comfortable advancing a molecule when we couldn’t confidently assess the potential risk.

That cautious approach becomes even more important in pediatric indications. Children are still developing, so you have to consider potential effects on developmental pathways and other biological processes that may not be relevant in adults. During toxicology studies, we're looking carefully to ensure the molecule doesn’t interfere with normal growth, neurological development, or reproductive biology. That means conducting studies in juvenile animals in addition to adults to identify any age-specific safety concerns.

For pediatric ASO development, potency is only one part of the equation. The goal is to find a molecule that combines strong efficacy, high specificity, and a safety profile appropriate for long-term use in children.

ARNA: Were there any assumptions that changed once you moved beyond early in vitro studies?

DW: One of the biggest assumptions was that strong in vitro efficacy would naturally translate into strong in vivo performance. That's the hope, but in reality there are many factors that influence whether that happens, including biodistribution, tissue penetration, and whether the ASO reaches the right regions of the brain at therapeutic levels.

Another assumption that changed involved pharmacokinetics. We initially expected the molecules to behave according to their plasma PK profile. Once they were cleared from circulation, we assumed their therapeutic effect would decline as well. What we’ve actually observed is that the ASOs can remain inside cells much longer than their circulating PK would suggest, allowing them to continue exerting their effect well after they've disappeared from the bloodstream.

We’re still investigating the mechanisms behind that prolonged cellular residence, but it has important implications for dosing strategies and treatment durability. It was a good reminder that pharmacokinetics alone doesn’t always tell the full story. Understanding how long these molecules remain active within their target cells is just as important as understanding how quickly they're cleared from the circulation.

ARNA: Rare disease programs often face limited models and patient data. How did those constraints influence your development strategy?

DW: Developing a therapy under these constraints is inherently challenging, but we were fortunate to have access to an excellent TUBB4A leukodystrophy mouse model developed at the Children’s Hospital of Philadelphia by Professor Vanderver's group. Because her team both treats patients and leads this laboratory research, there is a direct valuable connection between clinical observations and model data.

Our primary obstacle was species specificity: our lead ASO targets the human TUBB4A sequence, meaning it doesn't bind to the mouse gene. To establish proof of concept, we had to execute a dual-molecule parallel strategy that included:

• Efficacy (The Mouse Surrogate): We engineered a surrogate ASO specifically targeting the mouse TUBB4A gene. This surrogate generated highly encouraging efficacy data, demonstrating clear improvements in the disease phenotype.
• Safety & Activity (The Clinical Candidate): In parallel, we evaluated our actual human clinical candidate in non-human primates to characterize target knockdown and pharmacodynamic (PD) effects within the brain.

Because non-human primates don't have the disease, we couldn't assess functional outcomes like mobility improvements; those could only be evaluated using the surrogate in the mouse model. By comparing the PD data from the primate studies with the efficacy data from the mouse studies, we built a robust translational framework that gives us high confidence in how our clinical candidate will behave in humans.

ARNA: What translational signals gave you confidence the candidate could move toward the clinic?

DW: Two translational findings gave us the confidence to move the program toward the clinic.

The first was clear pharmacodynamic evidence of target engagement. We demonstrated that the ASO successfully reduced TUBB4A expression in the brain regions most relevant to the disease, confirming that the molecule was reaching its target and producing the intended biological effect.

The second was successful drug delivery. We're using the same intrathecal administration route in our preclinical studies that we plan to use clinically, delivering the ASO directly into the cerebrospinal fluid. One of the biggest challenges with antisense therapeutics is achieving sufficient distribution throughout the central nervous system, so it was encouraging to see the molecule reach key regions such as the basal ganglia and cerebellum.

Seeing both effective biodistribution and target knockdown in those regions gave us confidence that the therapeutic approach is clinically feasible. While we haven't yet completed our formal GLP toxicology studies, our dose-ranging and tolerability studies have been encouraging, with no significant safety concerns observed to date.

There's still important work ahead to fully characterize the safety profile, but the combination of robust target engagement, appropriate CNS distribution, and favorable early tolerability provides a strong foundation for advancing the program toward the clinic.

Closing Thoughts

TUBB4A leukodystrophy represents one of many rare neurogenetic diseases for which therapeutic options remain limited or nonexistent. For Williams and the SynaptixBio team, the challenge has been translating emerging biological insights into a development program capable of delivering a viable therapy.

While significant work remains before clinical evaluation, the program illustrates how advances in ASO technology are increasingly enabling developers to pursue diseases that previously lacked realistic therapeutic pathways. As the field continues to mature, the combination of disease-specific biology, targeted gene silencing, and translational rigor may provide new opportunities for addressing some of the most challenging unmet needs in pediatric neurology.

In Part 2 of this two-part series, we’ll discuss what it takes to build a lean ASO company, navigate regulatory uncertainty, and secure the resources necessary to advance a rare disease therapy into the clinic.

About The Expert

Dan Williams, Ph.D., is chief executive officer, executive director, and cofounder of SynaptixBio. Williams is an accomplished biopharmaceuticals leader with two decades of industrial experience from bench to operational and scientific executive roles. Throughout his career, Wiliams has served as vice president of research operations at Adaptimmune PLC and chief product officer at Meatable B.V.