Towards druggable targets in neurodevelopmental disorders: integrating single cell transcriptomics and reverse engineering in patient-derived cortical organoids
Progetto This project pursues the identification of functionally validated druggable targets for two neurodevelopmental disorders, Williams-Beuren and 7q11.23 microduplication syndromes (WBS and 7dup), whose genetic and phenotypic symmetry offers paradigmatic insight into the gene regulatory networks underlying intellectual disability and ASD. By using the latest available technologies for bulk and single cell RNAseq, proteomics, a recently developed platform of bioinformatic data deconvolution (reverse engineering of gene networks) and the most meaningful model for the study of human neurodevelopment, we expect to identify and validate for the first time a list of candidate genes susceptible of drug targeting with unprecedented reliability.
Aim 1: To define the phenotypic impact of 7q11.23 CNV, at the functional, transcriptomic and proteomic levels, in cortical neurons and astrocytes derived from human cortical organoids, consistent with the key role that transcriptional and translational dysregulation play in WBS and 7dup. Aim2: To dissect in a systematic manner, for the transcriptomic phenotypes of neurons and astrocytes uncovered in AIM 1, the contribution of 7q11.23 genes and their downstream effectors. Aim3: The definition of high-confidence druggable targets. Targets from Aim 2 predicted to be affected or counter-balanced by existing drugs will be selected for validation.
Our proposed experimental design integrates innovative approaches within a defined timeline based in two phases: In phase one, we will use a platform of differentiation of 3D cortical organoids from patient-derived iPSC, aiming to analyze mature stages at 100 and 200 days in vitro, in which organoids contain relevant cell populations, as well as evidence of primordial circuitry formation. We will profile the cortical organoids transcriptiomes by using the latest available technology in single cell RNAseq and in parallel we will obtain global proteomes. Their integrity will be established by electrophysiological properties and determination of cell type composition by quantitative tissue microarrays. In phase two we will establish high-confidence targets of validation by Reverse engineering of gene regulatory networks and then a multiplexed single-cell CRISPR screening (PerturbSeq) recently used for the functional interrogation of transcriptomes in mammalian cells.
From the preliminary result, and the degree of transcript protein concordance, we expect to prioritize 20-30 such key mediators. We will thus subject them, along with the 28 genes of the WBSCR to a thorough functional validation by performing CRISPR-based pooled screens, at the most informative time point in subset of lines of all genotypes. The goal is twofold: i) validate the molecular pathways inferred by reverse engineering; and ii) deconvolute their transcriptional impact to increase resolution of the gene regulatory network maps of the two cell types in the two disorders. The results obtained on this project entail not only a distinct set of high-confidence targets susceptible for drug modulation for these syndromes, but also would constitute a proof-of-principle approach that cements an strategy to tackle these kind of disorders that have proven so far resilient to intervention.