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.
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.
They treated neurons and 3D organoids differentiated from induced pluripotent stem cells (iPSC) of patients with TS with different ASO.
“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.
Using confocal microscopy, the team also confirmed that ASO treatment of transplanted TS organoids increased the amount of dendrite projections.
ABOVE: © Adventtr, iStock.
For neurons to function properly, calcium signaling is essential. Numerous neurological conditions are brought on by mutations that disrupt this process. The calcium voltage-gated channel subunit Alpha1 C (CACNA1C) gene point mutation that causes Timothy syndrome (TS) causes intracellular calcium levels to rise, delaying the closure of calcium channels and thereby enhancing neuron activation. Additionally, the mutation damages the dendritic projections of the neurons and prevents them from migrating during development.
Neuroscientist Sergiu Paşca of Stanford University studied antisense oligonucleotides as a treatment for this CACNA1C exon mutation and demonstrated that the treatment restored the CACNA1C channel’s function in a study published in Nature. 1 These results open the door to testing this strategy in clinical trials as a possible treatment for TS.
“We’ve been gradually understanding this disease better and better over the past 15 years,” Paşca said. He and his colleagues have recently explored this biology by creating three-dimensional brain organoids and models of neural stem cells from the neurons of TS patients. 2, 3.
He claimed that “a therapeutic opportunity just became a possibility because we suddenly had enough information about the biology of disease really by using these human stem cell models.”.
Paşca’s team investigates a TS type resulting from a mutation in exon eight A. Normal cell development involves a splicing mechanism that causes the cell to stop using this exon and favor exon eight. Neurones from patients carrying this mutation in CACNA1C, however, still utilize the mutated exon. In order to treat the dysregulated neuronal activity, Paşca’s team wondered if blocking the splicing of the mutated exon would promote the switch to the intact alternative variant.
Using short nucleotide sequences known as antisense oligonucleotides (ASO), the team investigated ways to prevent splicing at exon eight A by binding target RNA. They worked with neurons and three-dimensional organoids that were developed from TS patients’ induced pluripotent stem cells (iPSC) using various ASO. They discovered that multiple ASO lowered the expression of exon eight A through the use of qPCR and restriction fragment length polymorphism analysis.
“The ability to reduce the population’s representation of this eight A variant. The oligo therapy was really great, added Daniel Vogt, a developmental neuroscientist at Michigan State University who was not involved in the study. It proves that it’s achievable. “.
The scientists then looked into whether exon eight A’s decreased expression enhanced the CACNA1C calcium channel’s functionality. With the best performing ASO candidates, the team treated organoids derived from both normal and TS patient iPSC. They demonstrated that ASO reversed the delayed deactivation phenotype seen in the vehicle control-treated TS organoids using whole-cell patch clamping and calcium imaging.
The group created two separate brain organoids to test whether ASO corrected neuron migration. One of these organoids had migratory neurons that the scientists had labeled with GFP to monitor the cell’s motion. They used confocal microscopy to image the neuron migration both before and after ASO treatment. They cocultured these two types of organoids to create assembloids, which mimic various brain regions that communicate with one another. ASO treatment increased the efficiency of neuronal migration in TS assembloids, despite their lower migration efficiency when compared to neurons derived from normal cells.
Though exciting, Paşca clarified that these results were still obtained in a dish, so he and his colleagues used a transplantation model to investigate the impact of ASO in vivo. 4 Approximately 200 days after implanting cortical organoids from neurons of normal or TS patient neurons into neonatal rats, the team injected the animals with either saline or ASO.
Exon eight A was found to be expressed less frequently in rat brain tissues and implanted organoids following qPCR analysis by the team. ASO decreased intracellular calcium in cortical organoid neurons from TS iPSC after excitation, according to the team’s assessment of the amount of calcium inside cells following depolarization in the presence of a calcium indicator. The group also verified through confocal microscopy that the ASO treatment of transplanted TS organoids resulted in an increase in dendritic projections.
The team employed a variety of methods to bolster their findings, which impressed Mark Dell’Acqua, a neuroscientist at the University of Colorado who was not involved in the research. The human iPSC-derived neuron model was utilized extensively, specifically these organoids that were maintained in vitro instead of ex vivo, to investigate the possibility of reversing the genetic alteration through splicing modification using antisense oligonucleotides. “.
But he also noted that some of the neurological effects of TS may have been predetermined during embryonic development, and it’s unclear at this time whether this kind of treatment could make the impairments better in the past. He did, however, acknowledge that the study was a crucial beginning. According to Dell’Acqua, “it emphasizes an additional successful application of ASO technology to regulate splicing to correct a genetic disease.”
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