DELINEATING CELL TYPE-SPECIFIC<S>​</S> ROLES OF SETBP1 DURING NEURODEVELOPMENT USING HUMAN NEURAL ORGANOIDS AND TRANSCRIPTOMICS

Haploinsufficiency of the SETBP1 gene causes a highly heterogeneous neurodevelopmental syndrome (SETBP1-haploinsufficiency disorder) with the main phenotypic features including moderate-to-severe speech and language impairments, and wide variability in intellectual functioning. However, albeit known as a transcription factor and chromatin remodeller, the precise functions of SETBP1, encoding the SET-binding protein 1, during neurodevelopment are yet to be discovered. Here, we employed human induced pluripotent stem cell (iPSC)-derived neural organoids and transcriptomics to delineate the pathways regulated by SETBP1. We have generated SETBP1-knockout iPSCs with CRISPR/Cas9 gene-editing, followed by differentiation into self-patterned neural organoids. Morphological examination, transcriptomic profiling at whole organoid and single-cell levels, and protein abundance investigation were performed at two selected developmental timepoints. SETBP1-knockout organoids consistently showed gross morphological differences, rosette and transcriptomic anomalies during early organoid development, suggesting aberrations in cell fate commitment during embryonic brain development. Single cell transcriptomic and cell lineage analyses revealed a shift in abundance and development trajectory of forebrain progenitors and neurons. Cell-type specific gene ontology analyses demonstrated that dysregulated pathways were related to brain morphogenesis, cilia organisation, axon projection and synaptic function. Targeted modulation of developmental signalling alleviated morphological anomalies in SETBP1-knockout organoids. Our work is, to our knowledge, the first mechanistic investigation of the currently unknown neurodevelopmental roles of SETBP1 using human gene-edited iPSCs and neural organoids. Together, our findings demonstrate that SETBP1 is important for forebrain neuron differentiation. This work paves the way to gaining insights into the aetiological mechanisms that go awry in SETBP1-haploinsufficiency disorder.