Investigation of genetic determinants of Angelman Syndrome

ABSTRACT The Angelman Syndrome (AS) is a rare neurodevelopmental disorder caused by the loss of the maternally expressed UBE3A gene, which is located in the 15q11.2–q13 chromosomal region. AS can be caused by different genetic mechanisms, which all share the loss of the UBE3A gene. However, patients carrying chromosomal deletions show more severe clinical manifestations. This suggests the contributory role of co-deleted genes neighboring UBE3A to disease severity, which has never been dissected. This study aims to investigate the contribution of other genes lost in deletion patients to AS pathophysiology. In particular, we focus on the non-imprinted genes GABRB3, GABRA5, GABRG3, ATP10A, and HERC2, which are consistently lost in deletion-type AS independently from the extension of the chromosal deletion. Using genetically engineered human induced pluripotent stem cells (hiPSCs) differentiated into cortical neurons (iNeurons), we study the impact of AS genetic determinants in a human genetic context. To inactivate UBE3A and the other genes of interest, individually or in combination, we employ Prime Editing (PE), an advanced genome editing technology that allows to introduce targeted edits without generating double-strand DNA breaks. We first design engineered PE guide RNAs (epegRNAs) mediating the knockout of each gene of interest, clone PE-mediating plasmids containing each a specific epegRNA and successfully validate them on HEK293T cells using a competition-based PCR assay (cbPCR). We then generate vectors capable of mediating the simultaneous editing of multiple genes. hiPSCs are transfected with the first multiplex vector, which we are currently validating. Leveraging advanced genome editing, this study builds a novel framework for modeling deletion-type AS and lays the foundation for future studies aimed at molecularly dissecting the contribution of genetic determinants of AS pathogenesis in human neurons. We also expect that this battery of hiPSC lines will be an important tool for the scientific community working in the AS field.