Epilepsy‐associated <scp>SCN2A</scp> ‐ <scp>L1342P</scp> mutation drives network hyperexcitability and widespread transcriptomic changes in human cortical organoids

Abstract Objective SCN2A pathogenic mutations, such as the recurrent heterozygous Nav1.2‐L1342P, are monogenic causes of epilepsy. In this human‐induced pluripotent stem cell–derived model system, we aim to investigate the molecular and cellular mechanisms underlying SCN2A ‐L1342P‐associated pathology. Methods Using a human male induced pluripotent stem cell (iPSC) reference line (KOLF) carrying the Nav1.2‐L1342P mutation, we generated three‐dimensional (3D) cortical organoids for functional studies. Patch‐clamp, multi‐electrode array (MEA) recordings, immunocytochemistry, and RNA sequencing were used to characterize the disease phenotypes. Results Nav1.2‐L1342P organoid neurons displayed increased intrinsic excitability and amplified excitatory post‐synaptic currents, which are consistent with an increase in excitatory synapse formation revealed by SYN1/PSD95 immunostaining. Moreover, elevated network firing activity, as demonstrated by MEA, indicates a pronounced network hyperexcitability. Transcriptomic profiling of organoids carrying the Nav1.2‐L1342P mutation further revealed significant alterations in synaptic, glutamatergic, developmental, and senescence/apoptotic pathways. Significance Our findings demonstrate that the Nav1.2‐L1342P mutation drives a multifaceted disease phenotype, including network hyperexcitability and disruption of pathways related to neuronal and synaptic functions. These results advance our understanding of SCN2A ‐related developmental and epileptic encephalopathy (DEE), laying a foundation for personalized interventions.