Timothy syndrome (TS) is a severe, multisystem disorder characterized by autism, epilepsy, long-QT syndrome and other neuropsychiatric conditions¹. TS type 1 (TS1) is caused by a gain-of-function variant in the alternatively spliced and developmentally enriched CACNA1C exon 8A, as opposed to its counterpart exon 8. We previously uncovered several phenotypes in neurons derived from patients with TS1, including delayed channel inactivation, prolonged depolarization-induced calcium rise, impaired interneuron migration, activity-dependent dendrite retraction and an unanticipated persistent expression of exon 8A^2,3,4,5,6. We reasoned that switching CACNA1C exon utilization from 8A to 8 would represent a potential therapeutic strategy. Here we developed antisense oligonucleotides (ASOs) to effectively decrease the inclusion of exon 8A in human cells both in vitro and, following transplantation, in vivo. We discovered that the ASO-mediated switch from exon 8A to 8 robustly rescued defects in patient-derived cortical organoids and migration in forebrain assembloids. Leveraging a transplantation platform previously developed⁷, we found that a single intrathecal ASO administration rescued calcium changes and in vivo dendrite retraction of patient neurons, suggesting that suppression of CACNA1C exon 8A expression is a potential treatment for TS1. Broadly, these experiments illustrate how a multilevel, in vivo and in vitro stem cell model-based approach can identify strategies to reverse disease-relevant neural pathophysiology.

蒂莫西综合征 (TS) 是一种严重的多系统疾病，其特征为自闭症、癫痫、长 QT 综合征和其他神经精神疾病¹。 TS 1 型 (TS1) 是由选择性剪接和发育富集的CACNA1C外显子 8A中的功能获得性变异引起的，而不是其对应的外显子 8。我们之前发现了来自 TS1 患者的神经元的几种表型，包括延迟性通道失活、长时间去极化诱导的钙升高、中间神经元迁移受损、活动依赖性树突回缩以及外显子 8A ^2,3,4,5,6意外的持续表达。我们推断将CACNA1C外显子利用从 8A 转换为 8 将代表一种潜在的治疗策略。在此，我们开发了反义寡核苷酸 (ASO)，可在体外和移植后的体内有效减少人类细胞中外显子 8A 的包含。我们发现，ASO 介导的从外显子 8A 到 8 的转换有力地挽救了患者来源的皮质类器官的缺陷和前脑组合体的迁移。利用先前开发的移植平台⁷，我们发现单次鞘内 ASO 给药可挽救患者神经元的钙变化和体内树突回缩，这表明抑制CACNA1C外显子 8A 表达是 TS1 的潜在治疗方法。从广义上讲，这些实验说明了基于多层次、体内和体外干细胞模型的方法如何确定逆转疾病相关神经病理生理学的策略。