Fragile X syndrome (FXS) is the most common inherited form of intellectual disability and a leading genetic cause of autism spectrum disorders (ASD). FXS is caused by the loss of functional fragile X mental retardation protein (FMRP). Previous works have focused on the role of FMRP as a translational regulator, and many mRNA targets of FMRP have been shown to be ASD-linked genes. Despite major progress to characterize underlying disease mechanisms in animal models that has led to several clinical trials, including phase 3 clinical trials of drugs modulating metabotropic glutamate and GABA receptors, improvements of behavioral and cognitive outcomes in patients have unfortunately been largely unsuccessful. We believe that a major gap in the preclinical phase of drug development for FXS can be addressed by the development of human FXS induced pluripotent stem cell (iPSC) derived models, which will enable us to identify human specific therapeutic targets and evaluate novel therapeutic approaches. Human iPSCs are pluripotent and are able to generate many different cell types. Three-dimensional (3D) organoid culture of iPSCs has evolved from embryoid body culture, quite faithfully following human organogenesis, and provides a new platform to investigate human brain development in a dish, otherwise inaccessible to experimentation. We have developed FXS iPSC models, including 2D neural progenitor cells (NPCs)/cortical neurons and 3D cortical organoids, and identified a number of FMRP target mRNAs in the human context. Furthermore, we have observed abnormalities associated with the loss of FMRP at molecular, cellular and electrophysiological levels in FXS iPSC models. Intriguingly, our preliminary data suggest that PI3K inhibitors, but not mGluR5 antagonists, could rescue cellular phenotypes in human FXS iPSC derived model systems, potentially validating the failure of positive preclinical mouse studies with negative human trials. In this proposed study, we aim to use human specific iPSC models as translational tools to develop novel therapeutic approaches for FXS. First, we will determine the therapeutic effects of compounds targeting candidate pathways in FXS organoids (Aim 1). Second, we will develop CRISPR-based genomic and epigenomic editing therapeutic approaches to reactivate FMR1 expression in FXS organoids (Aim 2). Third, we will conduct molecular phenotype and FMR1-reactivation-based small molecule screens (Aim 3). Our proposed works will lead to the identification of novel therapeutic targets and the development of new treatment strategies for FXS.
