Calcium signaling is integral to proper neuron function. Mutations that impair this process cause a variety of neurological disorders. In Timothy syndrome (TS), a point mutation in the gene Calcium Voltage-Gated Channel Subunit Alpha1 C (CACNA1C) delays the closing of calcium channels, increasing intracellular calcium, which consequently enhances neuron activation. The mutation also hinders the migration of neurons during development and impairs their dendrite projections.
In a study published in Nature, Sergiu Paşca, a neuroscientist at Stanford University, investigated antisense oligonucleotides as an intervention for this CACNA1C exon mutation and showed that the treatment restored the function of the CACNA1C channel.1 These findings pave the way for taking this approach into clinical trials as a potential TS therapy.
“Over the past 15 years, we've been progressively understanding this disease better and better,” said Paşca. Recently, he and his team developed neuronal stem cell models and three-dimensional brain organoids from the neurons of patients with TS to explore this biology.2,3
“We just suddenly had enough information about the biology of disease really by using these human stem cell models that a therapeutic opportunity just became a possibility,” he said.
Paşca’s group studies a TS type caused by mutation in exon eight A. Normally, during development, cells stop using this exon and favor exon eight, in a process mediated by splicing. However, neurons from patients with this CACNA1C mutation continue to use the mutated exon. Paşca’s team considered whether inhibiting the splicing of the mutated exon would encourage the switch to the intact alternative variant and treat the dysregulated neuronal activity.
The team explored antisense oligonucleotides (ASO), which are short nucleotide sequences that bind target RNA, to inhibit splicing at exon eight A. They treated neurons and 3D organoids differentiated from induced pluripotent stem cells (iPSC) of patients with TS with different ASO. Using qPCR and restriction fragment length polymorphism analysis, they found that several ASO reduced the expression of exon eight A.
“The fact that they can make this eight A variant less representative in the population... with the oligo therapy, that was really nice,” said Daniel Vogt, a developmental neuroscientist at Michigan State University who was not involved in the research. “It shows that it's doable.”
Next, the researchers investigated if the reduced expression of exon eight A improved the function of the CACNA1C calcium channel. The team treated organoids derived from both normal iPSC and iPSC from TS patients with the highest performing ASO candidates. Using calcium imaging and whole-cell patch clamping, they showed that ASO reversed the delayed deactivation phenotype observed in the vehicle control-treated TS organoids.
To assess if ASO corrected neuron migration, the team generated two different brain organoids. One of these organoids included migrating neurons that the researchers labeled with GFP to track the cells’ movement. They cocultured these two types of organoids to form assembloids, which model different brain regions that communicate with each other, and imaged the neuron migration with confocal microscopy before and after ASO treatment. While neurons in TS assembloids migrated less efficiently compared to those derived from normal cells, ASO treatment restored normal migration activity.
Paşca explained that while these results were exciting, they were still all done in a dish, so he and his team turned to a transplantation model to study the effect of ASO in vivo.4 The team implanted cortical organoids from normal or TS patient neurons into neonatal rats and injected the animals with ASO or saline approximately 200 days later.
On performing qPCR, the team found that ASO decreased the expression of exon eight A in both rat brain tissues and tissues from the implanted organoids. When the team assessed the amount of calcium inside of cells after stimulating depolarization in the presence of a calcium indicator, they found that ASO lowered intracellular calcium in cortical organoid neurons from TS iPSC after excitation. Using confocal microscopy, the team also confirmed that ASO treatment of transplanted TS organoids increased the amount of dendrite projections.
Mark Dell’Acqua, a neuroscientist at the University of Colorado who was not affiliated with the study, was impressed with the number of approaches the team used to support their findings. “They really leveraged the human iPSC derived neuron model, in particular these organoids maintained in vitro slash ex vivo, to study how you could maybe reverse the genetic change using these antisense oligonucleotides by altering splicing.”
However, he pointed out that some of the neurological effects in TS could be set in place during embryonic development, and it’s not currently clear if this type of treatment could correct the impairments retroactively. Nonetheless, he said that the study was an important first step. “It emphasizes an additional successful application of ASO technology to regulate splicing to correct a genetic disease,” Dell’Acqua said.
- Chen X, et al. Antisense oligonucleotide therapeutic approach for Timothy syndrome. Nature. 2024;628:818-825
- Paşca SP, et al. Using iPSC-derived neurons to uncover cellular phenotypes associated with Timothy syndrome. Nat Med. 2011;17:1657-1662
- Birey F, et al. Assembly of functionally integrated human forebrain spheroids. Nature. 2017;545:54-59
- Revah O, et al. Maturation and circuit integration of transplanted human cortical organoids. Nature. 2022; 610:319-326
