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

BRITE-Eye：由高通量遗传筛选、人类神经元功能读数和机器学习驱动的中枢神经系统治疗靶点的集成发现引擎

• 批准号: 10699137
• 负责人: Steve John Ryan
• 金额: $ 172.19万
• 依托单位: QUELL TX, INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-19 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10699137
• 关键词: Action Potentials; Address; Affect; Architecture; Area; Behavior; Benchmarking; Biochemical; Biological Assay; Biology; Candidate Disease Gene; Cell model; Cells; Central Nervous System; Central Nervous System Agents; Central Nervous System Diseases; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Data; Data Set; Databases; Dimensions; Disease; Disease Pathway; Disease model; Down-Regulation; Drug Targeting; Electrophysiology (science); Epilepsy; Eye; FMR1; FMRP; Fingerprint; Fragile X Syndrome; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Screening; Genetic Transcription; Genomics; Guide RNA; Heterogeneity; Human; Human Genome; Individual; Induced pluripotent stem cell derived neurons; Industrialization; Intervention; Learning; Libraries; Light; Location; Machine Learning; Maps; Marketing; Measurement; Measures; Mediating; Medical; Modality; Modeling; Molecular; Molecular Target; Neurodevelopmental Disorder; Neurons; Optics; Patients; Pharmaceutical Preparations; Phase; Phenotype; Physiological; Physiology; Population; Predictive Value; Process; Resolution; Screening Result; Specificity; Synapses; System; Technology; Testing; Therapeutic; Training; Translating; Validation; Viral; Western Blotting; Work; analysis pipeline; analytical tool; automated analysis; convolutional neural network; deep learning; deep neural network; disease phenotype; drug discovery; excitatory neuron; functional genomics; gene function; genetic approach; genetic testing; genome wide screen; genome-wide; induced pluripotent stem cell; instrument; knock-down; loss of function; nervous system disorder; neurophysiology; new therapeutic target; novel; nuclease; particle; pharmacologic; programs; screening; success; therapeutic development; therapeutic gene; therapeutic target; therapeutically effective; transcriptomics; vector

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.

项目摘要 神经系统疾病会影响全球数百万患者，代表了主要的未满足医疗需求。最近的 与开发新的中枢神经系统（CNS）治疗的新类别相比 其他疾病区域。 CNS药物发现过程中的一个关键障碍是需要细胞模型， 测定和可以更可靠地评估与疾病相关的神经生理参数的技术 在单个神经元和突触的水平上的人类细胞环境，并具有捕获的量表和分辨率 这些系统的复杂性和可变性。我们建议通过整合三个 关键技术 - （i）人类神经元中全光生理学的高吞吐量Britetm平台，该平台 每天每天约500,000个神经元，达到单电池和单一电势分辨率 乐器; （ii）使用CRISPR核酸酶破坏基因功能的基因组筛选； （iii）机器学习 指纹的鉴定，代表具有单细胞分辨率的复杂生理表型。这 第二阶段计划包括四个关键目标。 1）在人类神经元中建立CRISPRN筛查条件。 我们将选择20个候选靶基因，包括癫痫和神经发育目标以进一步优化 测定条件与全光生理学表型兼容，包括遗传破坏的时间和 CRISPRN/GRNA成分的浓度有效地敲低基因靶标。 2）建立深度学习 - 用于单细胞表型的功率分析工具。我们将使用深层神经网络学习紧凑的矢量 药理干预后神经元行为的表示，利用了我们的单细胞分辨率 神经元种群中的测量和适应性潜在异质性。 3）识别遗传 使用全基因组CRISPRN屏幕的神经元功能调节剂。我们将结合实验 AIMS 1-2中建立的条件和分析模型，以执行全基因组CRISPRN屏幕（> 18,000 基因靶标）具有野生型人IPSC兴奋性神经元中的Arry GRNA库。我们将识别基因 下调下调的目标导致功能参数发生重大变化。潜在的命中和特异性 使用单个GRNA和QPCR和免疫印迹将在独立的回合中确认目标敲低 测定。 4）预测并验证脆弱X综合征的人IPSC-神经元模型中的表型救援。 最后，我们将评估AIM 3中开发的功能指纹的预测值，以生成A 我们在人类中确定的可以营救（抑制）表型参数的基因靶标的候选列表 神经发育障碍的细胞模型，脆弱的X综合征。我们将调节 这些潜在的遗传补充剂在FMR1-/Y IPSC-神经元和基准表型救援中使用CRISPRN 使用FMRP的遗传重新引入。成功完成拟议的工作有可能产生新的 了解人类神经生理学的分子结构和新型治疗平台 靶标识别集中于分子基础，以调节神经生理疾病机制。