INVESTIGATING GENETIC DETERMINANTS OF ANGELMAN SYNDROME IN HUMAN NEURONS

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by severe neurodevelopmental delay, absence of speech, intellectual disability, epilepsy, and sleep problems. There are no available treatments to prevent or modify the course of the disease. AS arises from diverse genetic causes, grouped into non-deletions and deletions of the 15q11-q13 region encompassing UBE3A, the main causal gene, and other genes. Deletion patients show more severe phenotypes, indicating that genes flanking UBE3A are important determinants of AS clinical manifestations. However, the cellular and molecular mechanisms underlying AS remain poorly understood. Using hiPSCs-derived human iNeurons, we employ CRISPR/Cas9 editing to generate lines mimicking the repertoire of AS genetic defects. To investigate the impact of UBE3A loss on human synaptic development, we generate UBE3A-KO hiPSCs-derived iNeurons and characterize them through optical microscopy, electrophysiology, and calcium imaging. UBE3A produces different splicing isoforms, which are differentially localized either in the nucleus or in the cytoplasm. Isoform-specific UBE3A-KO iNeurons are generated, providing an ideal platform to explore the poorly understood mechanisms governing isoform-specific localization and their relative contribution to neuronal function. To explore the contribution of additional genes lost in 15q11-q13 deletions, we leverage multiplex CRISPR-based editing to inactivate UBE3A and other genes, individually or in combinations, to establish genotype-phenotype correlations at the cellular and molecular levels. This project is expected to uncover novel pathogenic mechanisms of AS and how genetic heterogeneity could result in phenotypic differences among AS patients and might offer new perspectives for personalized medicine.