A Highthroughput Targeted Genetic Screen for Modulators of Nociception

伤害感受调节剂的高通量靶向遗传筛选

• 批准号: 9582710
• 负责人: AJAY K DHAKA
• 金额: $ 23.33万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2020-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9582710
• 关键词: Acute Pain; Address; Adverse effects; Affect; Animal Model; Behavior; Behavioral; Behavioral Assay; Biological Models; Biological Process; Biology; Brain; CRISPR/Cas technology; Calcium; Caliber; Candidate Disease Gene; Chemicals; Chronic; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Communities; Conscious; DNA Sequence Alteration; Data; Development; Economic Burden; Emotional; Ensure; Esthesia; Face; Gene Expression; Genes; Genetic; Genetic Determinism; Genetic Screening; Genetic Transcription; Head; Human; Image; Individual; Injury; Instruction; Intervention; Irritants; Knock-out; Label; Larva; Lead; Lesion; Mammals; Mediating; Medical; Methodology; Methods; Microscopy; Modality; Molecular; Mus; Mutation; Nerve Endings; Nervous system structure; Neuraxis; Neurons; Nociception; Nociceptive Stimulus; Nociceptors; Operative Surgical Procedures; Organism; Pain; Patients; Peripheral; Persistent pain; Pharmaceutical Preparations; Pharmacology; Phenotype; Play; Population; Productivity; RUNX3 gene; Reporter; Research; Resources; Rodent; Role; Skin; Societies; Source; Spinal Ganglia; Stimulus; Structure of trigeminal ganglion; System; Technology; Testing; Therapeutic Intervention; Time; Touch sensation; Transducers; Transgenic Organisms; United States; Vertebrates; Visual; Wages; Zebrafish; addiction; base; behavioral response; chronic pain; chronic painful condition; cost; disabling symptom; effective therapy; gene function; genome editing; in vivo; innovation; interest; knockout gene; loss of function; neural circuit; neurodevelopment; nociceptive response; novel; pain perception; pain sensation; response; sensor; somatosensory; targeted treatment; transcriptome sequencing

Among our senses nociception, the ability to detect noxious stimuli, is required for an organism's survival. Nociception induces the sensation of pain and prompts avoidance of the pain source so as to minimize injury. Noxious stimuli are detected by small diameter primary peripheral neurons (nociceptors) via nerve endings that project to the skin of the head and body, and this information is then transmitted to the brain, resulting in the conscious perception of pain. Debilitating chronic pain conditions affect hundreds of millions of people and impose a severe physical, emotional and economic burden on both individuals and society as a whole. Despite great advances, much remains to be understood about how painful stimuli are perceived and coded by the nervous system. This has resulted in a lack of effective therapies and methods to identify patients that respond to current treatments. Furthermore, currently available drug-based therapies have numerous deleterious side effects and/or potential for abuse and addiction, while not being effective for the treatment of chronic conditions. It is therefore vital to gain a more comprehensive understanding of the biology of these sensations, which could lead to the development of targeted treatment of chronic pain. Whole transcriptome sequencing has provided vast amounts of information about genes that are preferentially expressed in nociceptive neurons yet there have no practical or efficient methodologies to interrogate the role these genes play in nociception, as traditional approaches are slow, cumbersome and prohibitively expensive. Here we propose to use a relatively highthroughput CRISPR based genome editing strategy to visually and behaviorally probe the function of nociceptor enriched genes, utilizing the zebrafish model system. The zebrafish provides an intriguing model system to study nociception. The neural circuits underling nociception in zebrafish larvae are highly analogous to those found in higher vertebrates such as rodents and humans. Furthermore we've shown that zebrafish larvae have a functionally diverse peripheral and central nervous system and respond robustly to noxious stimuli. Additionally zebrafish can be generated in large numbers at low costs and their small size allows for rapid upscaling using existing high throughput platforms, which is not possible with other vertebrate systems such as rodents. Using CRISPR to knock out/down gene expression, our strategy allows us to assess the function of 4 genes per week. Gene knock out will occur in transgenic reporter lines that specifically label nociceptor populations allowing visual assessment of the role of any given gene in the development and targeting of these neurons. We will then use a robust larval locomotor behavioral assay to characterize the effects of genetic mutations on nociceptive response to touch, heat, cold and chemical irritants. Our preliminary data demonstrate that we can identify mutations affecting distinct pain modalities. We expect that this genetic screen will provide a resource for the community interested in the development of neural circuits and the perception of pain, and may provide targets for potential therapies for debilitating painful conditions.

在我们的感官中，伤害感受（即检测有害刺激的能力）是有机体生存所必需的。 伤害感受会引起疼痛感并促使回避疼痛源，以最大程度地减少伤害。 小直径初级周围神经元（伤害感受器）通过神经末梢检测到有害刺激 投射到头部和身体的皮肤，然后将这些信息传输到大脑，从而产生 对疼痛的有意识的感知。令人衰弱的慢性疼痛影响着数亿人 给个人和整个社会带来严重的身体、情感和经济负担。尽管 尽管已经取得了巨大的进步，但关于疼痛刺激是如何被感知和编码的，还有很多东西有待理解 神经系统。这导致缺乏有效的疗法和方法来识别有反应的患者 到目前的治疗方法。此外，目前可用的药物疗法有许多有害的一面 影响和/或滥用和成瘾的可能性，同时对治疗慢性病无效 状况。因此，更全面地了解这些感觉的生物学至关重要， 这可能会导致慢性疼痛靶向治疗的发展。全转录组测序 提供了大量有关在伤害性神经元中优先表达的基因的信息 然而，还没有实用或有效的方法来探究这些基因在伤害感受中发挥的作用，因为 传统方法缓慢、繁琐且昂贵。这里我们建议使用相对 基于高通量 CRISPR 的基因组编辑策略，以视觉和行为方式探测 利用斑马鱼模型系统富含伤害感受器的基因。斑马鱼提供了一个有趣的模型 研究伤害感受的系统。斑马鱼幼虫伤害感受的神经回路高度相似 那些在啮齿动物和人类等高等脊椎动物中发现的物质。此外，我们还表明斑马鱼 幼虫具有功能多样的外周和中枢神经系统，对有害物质反应强烈 刺激。此外，斑马鱼可以以低成本大量繁殖，而且它们的体型较小，因此可以 使用现有的高通量平台快速升级，这是其他脊椎动物系统不可能实现的 例如啮齿类动物。使用 CRISPR 敲除/降低基因表达，我们的策略使我们能够评估 每周 4 个基因的功能。基因敲除将发生在专门标记的转基因报告系中 伤害感受器群体允许对任何给定基因在发育和发育过程中的作用进行视觉评估 以这些神经元为目标。然后，我们将使用强大的幼虫运动行为测定来表征 基因突变对触摸、热、冷和化学刺激物的伤害性反应的影响。我们的初步 数据表明我们可以识别影响不同疼痛方式的突变。我们预计这种遗传 screen 将为对神经回路和神经回路发展感兴趣的社区提供资源 疼痛的感知，并可能为使人衰弱的疼痛状况的潜在疗法提供目标。