Administrative Supplement: Functional Assessment of Variants in Organisms of Research (FAVOR) - Profiling Canonical Human Genes and their Variants through Disease Model Phenotyping.

行政补充：研究有机体变异的功能评估（FAVOR）——通过疾病模型表型分析典型人类基因及其变异。

• 批准号: 10228504
• 负责人: Trisha Brock
• 金额: $ 12.49万
• 依托单位: NEMAMETRIX, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-10 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10228504
• 关键词: Acetylcholine; Administrative Supplement; Aldicarb; Animal Model; Animals; Antidotes; Atropine; Attenuated; Award; Behavior; Benign; Benzodiazepines; Biological Assay; Biological Process; Botox; Botulinum Toxin Type A; Caenorhabditis elegans; Carbamates; Chemicals; Cholinergic Receptors; Cleaved cell; Clinical; Code; Complement; Dangerousness; Detection; Development; Diagnosis; Diazepam; Disease model; Drug Screening; Electrophysiology (science); Enzyme Reactivation; Epilepsy; Exposure to; Funding; Future; Genes; Goals; Health; Human; Investigational Drugs; Libraries; Measurable; Measures; Methods; Morphology; Muscarinic Acetylcholine Receptor; National Human Genome Research Institute; Nematoda; Neurotoxins; Neurotransmitters; Organism; Organophosphates; Orthologous Gene; Paralysed; Parents; Pathogenicity; Patients; Pesticides; Pharmaceutical Preparations; Phenotype; Poisoning; Pralidoxime chloride; Proteins; Protocols documentation; Recording of previous events; Research; Route; SNAP receptor; Seizures; Small Business Innovation Research Grant; Symptoms; Synapses; Synaptic Vesicles; System; Therapeutic; Toxic effect; Transgenic Organisms; Translations; United States National Institutes of Health; Variant; Video Recording; Work; Zebrafish; base; cholinergic synapse; esterase; gamma-Aminobutyric Acid; genetic variant; inhibitor/antagonist; medical countermeasure; nerve agent; neuronal survival; neurotoxic; neurotoxicity; neurotransmission; neurotransmitter release; novel; parent project; postsynaptic; presynaptic; prevent; programs; receptor; response; restoration; screening; success; tool; transmission process; vesicular release

Summary This supplement funding request is relevant for the development of chemical medical countermeasures to neurotoxin exposure. Specifically, we use a humanized animal model to provide greater relevance and translation of drugs as antidotes to the neurotoxic effects of exposure to aldicarb, a pesticide which causes potentially lethal buildup of acetylcholine (ACh) at the synapse. In the parent SBIR award, we are creating a polygenic humanization of C. elegans wherein three human genes involved in vesicular release of ACh (SNAP25, STXBP1 and STX1A) replace their orthologous loci (ric-4, unc-18 and unc-64) in a single transgenic line. The resulting polygenic humanized animal will be used as a platform for functional categorization of pathogenic genetic variants when patient-derived variants are installed. As a supplementary project, the double-humanized hSTXBP1/hSTX1A humanized animals, which we have already created, and a tiple-humanized hSNAP25/hSTXBP1/hSTX1A will be used as a platform for discovery of drugs that act as antidotes to aldicarb neurotoxicity. The human proteins functionally replace the worm orthologs, restoring regulated neurotransmitter release from the synapse. Treatment with aldicarb inhibits synaptic ACh esterase (AChE) and leads to build up of ACh at cholinergic synapses. This paralytic effect of aldicarb is well documented in prior C. elegans work. Yet, most common antidotes to aldicarb work via a diverse set of mechanisms, which have not been well characterized in C. elegans. As a result, we focus on four distinct mechanisms: (1) acting upstream to decrease presynaptic release of ACh (botulinum toxin A - "Botox", (2) acting downstream to inhibit ACh receptors (atropine), (3) acting directly on the aldicarb-blocked AChE by promoting enzyme reactivation (pralidoxime), and (4) reducing GABA transmission which alleviates convulsive symptoms (diazepam). In this work to establish an aldicarb antidote discovery system, two aims are sought. The first aim will be to establish aldicarb paralytic activity that can be easily detected in the humanized animal models. The second aim will be to measure the antidote effects of four drugs (atropine, pralidoxime, diazepam and Botox) on aldicarb-induced paralysis. Success of the project occurs when an assay protocol is found capable of detecting the effects of an antidote in reducing aldicarb toxicity. In future work, this humanized system can be used as a screening platform for aldicarb antidotes as well as a broad range of neurotoxic agents that threaten human health.

概括 该补充资金申请与化学医疗对策的开发有关 神经毒素暴露。具体来说，我们使用人性化动物模型来提供更大的相关性和 将药物转化为接触涕灭威（一种农药，可导致神经中毒）的解毒剂 突触处乙酰胆碱 (ACh) 的积累可能致命。在母公司 SBIR 奖项中，我们正在创建一个 秀丽隐杆线虫的多基因人源化，其中三个人类基因参与乙酰胆碱的囊泡释放 （SNAP25、STXBP1 和 STX1A）在单个转基因中替换其直系同源基因座（ric-4、unc-18 和 unc-64） 线。由此产生的多基因人源化动物将用作功能分类的平台 当安装源自患者的变异时，会产生致病性遗传变异。作为一个补充项目， 我们已经创建了双人源化 hSTXBP1/hSTX1A 人源化动物，以及 三重人源化 hSNAP25/hSTXBP1/hSTX1A 将用作发现药物的平台 涕灭威神经毒性的解毒剂。人类蛋白质在功能上取代了蠕虫直系同源物，恢复了 调节突触释放的神经递质。涕灭威治疗可抑制突触乙酰胆碱酯酶 (AChE) 并导致胆碱能突触处乙酰胆碱的积累。涕灭威的这种麻痹作用很好 记录在先前的秀丽隐杆线虫工作中。然而，最常见的涕灭威解毒剂通过多种不同的方式发挥作用。 机制，但尚未在秀丽隐杆线虫中得到很好的表征。因此，我们重点关注四个不同的领域 机制：(1) 作用于上游，减少突触前乙酰胆碱的释放（肉毒杆菌毒素 A - “Botox”，(2) 作用于下游抑制乙酰胆碱受体（阿托品），(3) 直接作用于涕灭威阻断的乙酰胆碱酯酶 促进酶重新激活（解磷定），以及 (4) 减少 GABA 传输，从而缓解惊厥 症状（地西泮）。在这项建立涕灭威解毒剂发现系统的工作中，我们追求两个目标。 第一个目标是建立可在人源化动物中轻松检测到的涕灭威麻痹活性 模型。第二个目标是测量四种药物（阿托品、解磷定、地西泮）的解毒作用 和肉毒杆菌）对涕灭威引起的麻痹。当找到检测方案时，项目就成功了 能够检测解毒剂降低涕灭威毒性的效果。在今后的工作中，这种人性化 系统可用作涕灭威解毒剂以及广泛的神经毒性药物的筛选平台 威胁人类健康的媒介。

项目成果

Phenotypic screening models for rapid diagnosis of genetic variants and discovery of personalized therapeutics.

• DOI: 10.1016/j.mam.2022.101153
• 发表时间: 2023-06
• 期刊: MOLECULAR ASPECTS OF MEDICINE
• 影响因子: 10.6
• 作者: Hopkins, Christopher E.;Brock, Trisha;Caulfield, Thomas R.;Bainbridge, Matthew
• 通讯作者: Bainbridge, Matthew