DECIPHERING REGULATORY MECHANSIMS DRIVING DRAVET SYNDROME IN BRAIN ORGANOIDS

Dravet syndrome (DS) is one of the most common developmental epileptic encephalopathies (DEEs), characterized by early onset epilepsy and severe neurodevelopmental impairment. Brain organoids enable the recapitulation of developmental processes, molecular mechanisms underlying cell differentiation and mimic the spatial organization and structural architecture of the developing human brain. Furthermore, assembloids, created by integrating different types of organoids, allow us to study interactions driven by distinct genotypes, offering a more comprehensive model of brain development and disease mechanisms. In this project, we aim to understand potential defects in excitatory/inhibitory imbalance and neuronal migration in Dravet syndrome compared to control organoid models. To achieve this, we developed organoids and assembloids at different time points using unguided protocols (Lancaster (2014)) to explore how the cell environment influences chromatin accessibility and epigenetic regulation. Brain organoids and assembloids were generated from Dravet induced pluripotent stem cell line (iPSCs) and human control embryonic stem cell line (H9 line) with cherry reporter. We obtain (H9::mCherry), Dravet organoids and assembloids (H9::mcherry+DS) at day 90 and dissected them in three pieces obtaining three different regions: the DS14 derived region, the intersection region and the H9::mCherry enriched region. Chromatin accessibility was profiled using ATAC-seq and compared across organoids and assembloid regions. Interestingly, the intersection region clustered more closely with DS14 genotype than with H9 genotype. These findings suggest that the DS genotype may exert a dominant, non–cell-autonomous influence on the surrounding cellular environment. Further analyses are required to elucidate the molecular mechanisms underlying this effect.