BRITE-Eye: An integrated discovery engine for CNS therapeutic targets driven by high throughput genetic screens, functional readouts in human neurons, and machine learning

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Project Summary Neurological disorders affect millions of patients worldwide and represent a major unmet medical need. Recent progress on developing new classes of central nervous system (CNS) therapeutics has lagged compared to other disease areas. A key obstacle in the CNS drug discovery process has been a need for cellular models, assays, and technologies that can more reliably assess disease-relevant neurophysiological parameters in a human cellular context at the level of individual neurons and synapses, with the scale and resolution to capture the complexity and variability of these systems. We propose to address this need through the integration of three key technologies – (i) our high throughput BRITETM platform for all-optical physiology in human neurons, which achieves single-cell and single-action-potential resolution with a throughput of ~500,000 neurons per day per instrument; (ii) genomic screens using CRISPR nuclease to disrupt gene function; (iii) machine learning for identification of fingerprints that represent complex physiological phenotypes with single-cell resolution. This Phase II program includes four key objectives. 1) Establish CRISPRn screening conditions in human neurons. We will select 20 candidate target genes, including epilepsy and neurodevelopmental targets to further optimize assay conditions compatible with all-optical physiology phenotyping, including timing of genetic disruption and concentration of CRISPRn/gRNA components for effective knockdown of gene targets. 2) Build deep-learning- powered analytical tools for single-cell phenotyping. We will use deep neural networks to learn a compact vector representation of neuronal behavior after pharmacological intervention that leverages our single cell resolution measurements and accommodates potential heterogeneity in the population of neurons. 3) Identify genetic modulators of neuronal function using a genome-wide CRISPRn screen. We will combine experimental conditions and analytical models established in Aims 1-2 to carry out a genome-wide CRISPRn screen (>18,000 gene targets) with arrayed gRNA libraries in wild-type human iPSC-excitatory neurons. We will identify gene targets whose downregulation leads to significant changes in functional parameters. Potential hits and specificity of target knockdown will be confirmed in independent rounds using single gRNA and qPCR and immunoblotting assays. 4) Predict and validate phenotypic rescue in a human iPSC-neuronal model of Fragile X Syndrome. Finally, we will assess the predictive value of the functional fingerprints developed in Aim 3 to generate a candidate list of gene targets that can rescue (suppress) phenotypic parameters we have identified in a human cellular model of the neurodevelopmental disorder, Fragile X syndrome. We will modulate the expression of these potential genetic suppressors with CRISPRn in FMR1-/y iPSC-neurons and benchmark phenotypic rescue using genetic re-introduction of FMRP. Successful completion of the proposed work has potential to yield a new understanding of the molecular architecture of human neurophysiology and a platform for novel therapeutic target identification focused on the molecular basis for modulation of neurophysiological disease mechanisms.