Abstracts

Rationale: Focal malformations of cortical development (FMCD), including focal cortical dysplasia (FCD), hemimegalencephaly (HME), and tuberous sclerosis (TSC), are significant contributors to medically intractable epilepsy. Brain-only mutations (so-called ‘brain somatic mosaicism') causing the aberrant activation of mTOR signaling have been identified as a major genetic cause of FCD and HME, classifying them as mTORopathies. Epilepsy surgery is the only treatment option for FCD and HME with intractable seizures. In TSC, everolimus, the approved mTOR inhibitor drug, shows limited efficacy for controlling seizures with notable adverse events such as immunosuppression and increased risk of infections. Thus, there is a need for more efficient and safer therapeutic options targeting MTOR in the brain. This study explores the use of antisense oligonucleotide (ASO) technology to target and modulate mTOR signaling in the brain as a potential therapeutic strategy for mTORopathies with potentially fewer adverse events.




Methods: We employed mouse FCDII models with somatic mosaicism to evaluate the efficacy of mouse Mtor-targeting ASOs. Following this, we screened human MTOR-targeting ASOs both in vitro, using human cell lines and brain organoids, and in vivo, including rodents and nonhuman primates (NHP). The lead ASOs were identified based on their ability to decrease MTOR levels and their safety when injected. Further, we assessed the therapeutic potential of the ASO in human embryonic stem cell-derived neurons with MTOR mutations, monitoring its impact on MTOR levels and its ability to rescue FMCD-related pathophysiological symptoms.




Results: In the FCDII mouse model, mouse Mtor-targeting ASOs significantly suppressed spontaneous seizures, with effects lasting approximately 6-8 weeks after a single injection, indicating a long-duration efficacy. Human MTOR-targeting ASOs screened in vitro showed a dose-dependent reduction in MTOR mRNA levels, and in vivo studies in NHPs confirmed these findings, with the lead ASO effectively reducing both MTOR mRNA and protein levels. Additionally, the MTOR-targeting ASOs ameliorated pathophysiological symptoms associated with FMCDs in human embryonic stem cell-derived neurons carrying MTOR mutations. Notably, the intrathecal administration of ASOs demonstrated a lower probability of adverse events compared to systemic everolimus treatment, reducing the risk of adverse events.




Conclusions: mTOR-targeting ASOs significantly reduce seizure activity in model mice, have long-lasting effects, and lower the risk of adverse events compared to everolimus. The intrathecal delivery of MTOR-targeting ASOs could offer a safer and more effective treatment for epilepsy in mTORopathies, showing promise as a novel therapeutic approach.




Funding: This research was funded by Sovargen Co., Ltd. This research was also supported by Korea Drug Development Fund funded by Ministry of Science and ICT, Ministry of Trade, Industry, and Energy, and Ministry of Health and Welfare (RS-2023-00282544, Republic of Korea).