Generation of isogenic human models of SYNGAP1 haploinsufficiency
1 Institute of Reconstructive Neurobiology, LIFE & BRAIN Center, University of Bonn School of Medicine & University Hospital Bonn, Germany
SYNGAP1 syndrome is a rare congenital neurodevelopmental disorder characterized by intellectual disability and epilepsy. It is caused by a heterozygous mutation in the SYNGAP1 gene that encodes for the ‘Synaptic Ras/Rap GTPase-activating’ (SynGAP) protein which is mainly restricted to the postsynaptic density of forebrain excitatory synapses. Here, we aim to assess the impact of SYNGAP1 haploinsufficiency on early human neural development and synapse formation. To this end, we are generating human pluripotent stem cells (hPSCs) with heterozygous SYNGAP1 knockout alleles for the subsequent derivation of standardized 2D neuronal cultures as well as cerebral organoids. Controlled derivation of excitatory or inhibitory neurons for 2D cultures is achieved via transcription factor-mediated forward programming employing doxycycline-inducible NGN2 or ASCL1/DLX2 transgenes integrated into the AAVS1 ‘safe-harbor’ locus. Within the initiation phase of this project we have successfully generated inducible NGN2- as well as ASCL1/DLX2-transgenic PSC lines including isogenic SYNGAP+/- cloneswhich are currently subjected to neuronal differentiation for subsequent phenotypic characterization. These studies include expression analyses of SynGAP1 as well as other pre- and postsynaptic proteins across in vitro differentiation in order to identify the optimal maturation level and time window for subsequent assays that will focus onsynapse formation, spine morphology and neuronal network activity.We expect our isogenic hPSC model for SYNGAP1 haploinsufficiencyto provide a valuable resource for deciphering cellular and molecular pathomechanisms leading to SYNGAP1 syndrome and to enable cell-based evaluation of candidate drugs counteracting SYNGAP1 pathophenotypes.