ESTABLISHMENT OF HIPSC-BASED BLOOD-BRAIN BARRIER <EM>IN VITRO </EM>MODELS OF RARE NEURODEVELOPMENTAL DISORDERS <EM> </EM>

Rare neurodevelopmental disorders such as SYNGAP1-related disorder (SYNGAP1), Cardio-Facio-Cutaneous syndrome (CFCS), and Costello syndrome (CS) remain poorly understood, and a potential involvement or contribution of Blood-Brain Barrier (BBB) (dys)function in these diseases has not investigated yet. This study aimed to establish in vitro BBB models to investigate disease-specific characteristics and evaluate novel drug delivery approaches. BBB models of SYNGAP1, CFCS, and CS were established using patient-derived or genetically engineered human induced pluripotent stem cells (hiPSCs) differentiated into brain capillary endothelial-like cells (BCELCs). Barrier integrity was assessed by transendothelial electrical resistance (TEER) and paracellular marker permeability measurements. Cellular and molecular characteristics were evaluated by immunofluorescence microscopy, high-throughput qPCR, and RNA sequencing. The role of SYNGAP1 in BBB development was studied in more detail in co-culture setups with isogenic microenvironmental cells and by single-cell RNA sequencing (scRNASeq). SYNGAP1, CFC, and CS BBB models exhibited functional barrier formation (TEER >1000 Ohms*cm²) and BBB-relevant marker expression (Claudin-5, ZO-1, Occludin, VE-cadherin, PECAM-1, GLUT1). Analyses of the SYNGAP1^-/- BBB model revealed a delayed barrier formation, significantly reduced TEER (~ 56%), and increased permeability (~ 80%) compared to the SYNGAP1^WT model. Additionally, scRNASeq results and co-culture experiments suggested a difference in the composition of the microenvironmental cells and their influence on barrier formation during SYNGAP1^-/- BCELC differentiation, indicating a potential role of SYNGAP1 in BBB development. In conclusion, the established BBB models of SYNGAP1, CFCS, and CS provide a valuable tool for studying disease mechanisms and a platform for testing and optimizing drug delivery into the brain.