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

摘要 这项补充资金申请与开发化学医学对策有关，以 暴露于神经毒素。具体地说，我们使用人性化的动物模型来提供更大的相关性和 将药物翻译成解毒剂，以缓解因接触敌百威而产生的神经毒性作用。 可能致命的乙酰胆碱(ACh)在突触上的积聚。在父SBIR奖中，我们正在创建一个 线虫囊泡释放三个人类基因的多基因人源化 (SNAP25、STXBP1和STX1A)在单个转基因中替换它们的直系基因座(RIC-4、UNC-18和UNC- 排队。由此产生的多基因人源化动物将被用作功能分类的平台 安装患者衍生的变异时的致病遗传变异。作为一个补充项目， 双人化hSTXBP1/hSTX1A人性化动物，我们已经创造了，和一个 三重人源化hSNAP25/hSTXBP1/hSTX1A将作为发现具有以下作用的药物的平台 敌百威神经毒性的解毒剂。人类蛋白质在功能上取代了蠕虫的同源物，恢复了 受调节的神经递质从突触释放。敌百威对突触ACh酯酶的抑制作用 (AChE)，并导致胆碱能突触上ACh的积聚。敌百威的这种麻痹作用很好。 在以前的线虫工作中被记录下来。然而，最常见的杀菌威解毒剂是通过一系列不同的 机制，这在线虫中还没有很好的特征。因此，我们专注于四个不同的 机制：(1)作用于上游以减少突触前ACh(肉毒杆菌毒素A-“肉毒杆菌”)的释放，(2) 作用于下游以抑制ACh受体(阿托品)，(3)直接作用于敌百威阻断的AChE 促进酶的重新激活(解磷定)，以及(4)减少GABA的传递，从而减轻惊厥 症状(安定)。在这项建立敌百威解毒剂发现系统的工作中，寻求两个目标。 第一个目标将是建立可以在人源化动物身上很容易检测到的敌百威麻痹活性。 模特们。第二个目标是测量四种药物(阿托品、解磷定、安定)的解毒效果 和肉毒杆菌素)对敌百威引起的麻痹。当找到一种分析方案时，项目就成功了 能够检测解毒剂在降低敌百威毒性方面的效果。在今后的工作中，这是人性化的 该系统可作为敌百威解毒剂以及多种神经毒性药物的筛选平台 威胁人类健康的毒剂。

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.