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ZEBRAFISH MODELS FOR DRAVET SYNDROME RESEARCH AND DISCOVERY

用于 Dravet 综合征研究和发现的斑马鱼模型

基本信息

批准号：
10543132
负责人：
Scott C Baraban
金额：
$ 51.03万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-01 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10543132
关键词：
Agonist Award Behavior Behavioral Benchmarking Biological Assay Blinded Brain CRISPR/Cas technology Calcium Cells Child Clinical Colorado Communities Convulsants Development Disease Drug resistance Electrodes Electrophysiology (science)Elements Epilepsy Etiology Exhibits Failure Family Functional disorder Gene Mutation Generations Genetic Goals Heterozygote Human Image Imaging Techniques Impairment Investigation Larva Libraries Locomotion Metabolic dysfunction Microfluidics Modeling Mutation National Institute of Neurological Disorders and Stroke Patients Pharmaceutical Chemistry Pharmaceutical Preparations Pharmacology Pharmacology Study Phenotype Research Resistance Resolution SCN1A protein Seizures Serotonin Signal Pathway Social Development Sodium Channel Techniques Technology Time Transgenic Organisms Work Zebrafish analog analysis pipeline blind calcium indicator childhood epilepsy comorbidity confocal imaging de novo mutation design dravet syndrome drug candidate drug development drug discovery drug use screening effective therapy excitatory neuron genome editing high risk high-throughput drug screening improved in vivo inhibitory neuron insight kinase inhibitor loss of function manufacture multidisciplinary mutant neural network novel preclinical development preclinical study receptor research and development screening severe intellectual disability side effect sudden unexpected death in epilepsy tool voltage

项目摘要

Project Summary/Abstract Dravet syndrome (DS), a catastrophic childhood epilepsy, is associated with severe intellectual disability, impaired social development, persistent drug-resistant seizures and a high risk of sudden unexpected death in epilepsy. Our recent investigation of zebrafish mutants featuring a loss-of-function sodium channel (scn1a) mutation (e.g., a gene mutation identified in ~80% of DS patients) focused on drug discovery and development, metabolic dysfunction and behavioral comorbidities. Using a high-throughput phenotype-based screening strategy and medicinal chemistry, we screened nearly 3000 drugs, successfully identified a serotonin (5HT) receptor mechanism underlying anti-seizure activity of clemizole and developed three novel clemizole analogs. Using CRISPR/Cas9 genome editing technology we generated new zebrafish mutant lines for chd2, gabrb3, pcdh19 and stxbp1 (e.g., de novo mutations seen in ~20% of DS patients). Interestingly, stxbp1 and gabrb3 mutants exhibit epileptic phenotypes. We also designed and manufactured a microfluidic, multi-channel electrode-integrated platform for long-term non-invasive electrophysiology on larval zebrafish (Hong et al. 2016) and developed a calcium imaging-analysis pipeline for studying seizure macro- and micro- networks in vivo (Liu and Baraban 2019). The proposed work will leverage these unique tools. Three specific aims are proposed: (i) to resolve neural networks responsible for seizures in larval DS zebrafish, (ii) to perform high-throughput drug screening using zebrafish and (iii) to further evaluate clemizole and related anti-seizure compounds. Techniques will include automated locomotion tracking, in vivo zebrafish electrophysiology, pharmacology, and fast calcium imaging using genetically encoded calcium- and voltage-sensitive indicators. Our results promise to simultaneously advance our long-term goals (i) to better understand the pathophysiology of genetic epilepsies and (ii) identify promising new treatment options for these intractable conditions.

项目概要/摘要 Dravet 综合征 (DS) 是一种灾难性的儿童癫痫症，与严重的智力障碍有关，社会发展受损、持续耐药性癫痫发作以及突然意外死亡的高风险癫痫。我们最近对钠通道（scn1a）功能丧失的斑马鱼突变体的研究突变（例如，在约 80% 的 DS 患者中发现的基因突变）专注于药物发现和发育、代谢功能障碍和行为合并症。使用基于高通量表型的筛选策略和药物化学，我们筛选了近3000个药物，成功鉴定出一个克立咪唑抗癫痫活性背后的血清素（5HT）受体机制，并开发了三种新型克立咪唑类似物。利用 CRISPR/Cas9 基因组编辑技术，我们生成了新的斑马鱼突变系 chd2、gabrb3、pcdh19 和 stxbp1（例如，约 20% 的 DS 患者中出现新生突变）。有趣的是， stxbp1 和 gabrb3 突变体表现出癫痫表型。我们还设计和制造了微流体、斑马鱼幼体长期无创电生理学多通道电极集成平台（Hong 等人，2016）并开发了一种钙成像分析管道，用于研究癫痫发作的宏观和微观体内网络（Liu 和 Baraban 2019）。拟议的工作将利用这些独特的工具。三具体提出的目标是：(i) 解决 DS 斑马鱼幼虫癫痫发作的神经网络问题，(ii) 执行使用斑马鱼进行高通量药物筛选，以及 (iii) 进一步评估克立咪唑和相关的抗癫痫药物化合物。技术将包括自动运动跟踪、体内斑马鱼电生理学、药理学，以及使用基因编码的钙和电压敏感指示剂的快速钙成像。我们的结果有望同时推进我们的长期目标 (i) 更好地了解遗传性癫痫的病理生理学，以及（ii）为这些棘手的问题确定有希望的新治疗方案状况。