Manipulating microRNA-target interactions to rescue protein levels in haploinsufficient disorders

Project Summary/Abstract: Correcting gene dosage is crucial for addressing neurodevelopmental disorders (NDDs) linked to haploinsufficient genes, where the loss of one allele disrupts normal gene function. This research focuses on developing a cutting-edge platform to identify and manipulate microRNA (miRNA)-mediated repression of haploinsufficient genes, a strategy that has been underexplored due to technical limitations. Our approach integrates advanced tools to map miRNA-target interactions (MTIs), block these interactions, and measure the ability of blocking specific MTIs to restore levels of protein translation with high precision and at scale. We will utilize AGO2 eCLIPseq to map cell-type and human-specific MTIs in iPSC-derived cortical neurons, providing a comprehensive view of target sites on haploinsufficient genes where miRNAs repress translation. To address the challenge of blocking specific MTIs with high throughput, we will employ a novel CRISPR-based system using pooled barcoded guide RNAs (gRNAs) that target and inhibit specific MTIs. This method, demonstrated to be effective in preliminary tests, will be adapted for pooled screening. We will combine the above techniques with single-cell Ribo-STAMP technology to quantify translational changes in target proteins at single-cell resolution, a significant advancement over current methods. This platform will first be validated in mouse cortical neurons before transitioning to human iPSC-derived neurons, where we aim to discover and validate promising MTI targets with potential to rescue protein levels in human haploinsufficient genes. Our innovative approach aims to map, block, and validate MTIs that can be targeted to rescue protein levels in haploinsufficient NDD causing genes. By systematically identifying and blocking these MTIs, we seek to establish a robust screening platform that could lead to new therapeutic strategies for NDDs and other haploinsufficient disorders. The successful execution of this project will provide critical insights into miRNA-mediated regulation of gene expression and offer potential novel avenues for clinical intervention.