Timothy syndrome (TS) is a severe, multisystem disorder characterized by autism, epilepsy, long-QT syndrome, and other neuropsychiatric conditions. 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. Researchers developed antisense oligonucleotides (ASOs) to decrease the inclusion of exon 8A in human cells, both in vitro and in vivo. The ASOs effectively rescued defects in patient-derived cortical organoids and migration in forebrain assembloids. In a transplantation model, 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. These experiments demonstrate how a multilevel, in vivo and in vitro stem cell model-based approach can identify strategies to reverse disease-relevant neural pathophysiology. The study highlights the potential of ASOs in treating TS by targeting the splicing of CACNA1C, which is crucial for neuropsychiatric conditions. The findings suggest that ASOs can modulate splicing of human CACNA1C both in vitro and in vivo, thereby rescuing molecular and cellular phenotypes of TS1. The study also shows that ASOs can be delivered in vivo using a organoid transplantation platform, and that they can rescue splicing and intracellular calcium flux defects in human neurons integrated into the rat cerebral cortex. The research provides a potential therapeutic strategy for a severe neurodevelopmental disorder caused by a single nucleotide variant in an alternatively spliced exon. The study demonstrates that ASOs can effectively modulate splicing in TS to reduce exon 8A without changing the overall level of CaV1.2 protein. The results indicate that ASOs can rescue ion flux kinetics, calcium dynamics, and associated cellular movement defects in TS. The study also shows that ASOs can be delivered in vivo and rescue TS-related phenotypes in transplanted human TS cells. The research provides a proof-of-concept for using ASOs in treating TS, highlighting their potential in neuropsychiatric disorders. The study underscores the importance of splicing regulation in CACNA1C and the potential of ASOs in targeting this process for therapeutic intervention. The findings suggest that ASOs can be a promising treatment for TS by correcting the splicing of CACNA1C and restoring normal channel function. The study also highlights the need for further research to refine ASO specificity and evaluate their long-term safety and efficacy in vivo.Timothy syndrome (TS) is a severe, multisystem disorder characterized by autism, epilepsy, long-QT syndrome, and other neuropsychiatric conditions. 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. Researchers developed antisense oligonucleotides (ASOs) to decrease the inclusion of exon 8A in human cells, both in vitro and in vivo. The ASOs effectively rescued defects in patient-derived cortical organoids and migration in forebrain assembloids. In a transplantation model, 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. These experiments demonstrate how a multilevel, in vivo and in vitro stem cell model-based approach can identify strategies to reverse disease-relevant neural pathophysiology. The study highlights the potential of ASOs in treating TS by targeting the splicing of CACNA1C, which is crucial for neuropsychiatric conditions. The findings suggest that ASOs can modulate splicing of human CACNA1C both in vitro and in vivo, thereby rescuing molecular and cellular phenotypes of TS1. The study also shows that ASOs can be delivered in vivo using a organoid transplantation platform, and that they can rescue splicing and intracellular calcium flux defects in human neurons integrated into the rat cerebral cortex. The research provides a potential therapeutic strategy for a severe neurodevelopmental disorder caused by a single nucleotide variant in an alternatively spliced exon. The study demonstrates that ASOs can effectively modulate splicing in TS to reduce exon 8A without changing the overall level of CaV1.2 protein. The results indicate that ASOs can rescue ion flux kinetics, calcium dynamics, and associated cellular movement defects in TS. The study also shows that ASOs can be delivered in vivo and rescue TS-related phenotypes in transplanted human TS cells. The research provides a proof-of-concept for using ASOs in treating TS, highlighting their potential in neuropsychiatric disorders. The study underscores the importance of splicing regulation in CACNA1C and the potential of ASOs in targeting this process for therapeutic intervention. The findings suggest that ASOs can be a promising treatment for TS by correcting the splicing of CACNA1C and restoring normal channel function. The study also highlights the need for further research to refine ASO specificity and evaluate their long-term safety and efficacy in vivo.