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

总结 这一补充资金要求与化学医学对策的发展有关， 神经毒素暴露具体而言，我们使用人源化动物模型来提供更大的相关性， 涕灭威是一种农药， 突触处乙酰胆碱（ACh）的潜在致命积聚。在母公司SBIR奖中，我们正在创建一个 多基因人源化C.其中三个人类基因参与ACh的囊泡释放 在单个转基因植物中，SNAP 25、STXBP 1和STX 1A取代了它们的正向同源基因座（ric-4、unc-18和unc-64 线所得的多基因人源化动物将被用作功能分类的平台。 致病性遗传变异时，患者衍生的变种安装。作为补充项目， 双重人源化hSTXBP 1/hSTX 1A人源化动物，我们已经创造了，和一个人源化动物。 三重人源化hSNAP 25/hSTXBP 1/hSTX 1A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的直系同源物， 调节神经递质从突触释放。涕灭威处理抑制突触乙酰胆碱酯酶 （乙酰胆碱酯酶），并导致建立乙酰胆碱在胆碱能突触。涕灭威的这种麻痹作用 记录在之前的C。优雅的工作。然而，涕灭威最常见的解毒剂是通过一系列不同的 机制，这还没有得到很好的特点，在C。优雅因此，我们专注于四个不同的 机制：（1）作用于上游以减少ACh（肉毒杆菌毒素A -“Botox”，（2） 作用于下游以抑制ACh受体（阿托品），（3）通过以下途径直接作用于涕灭威阻断的AChE： 促进酶的再活化（解磷定），和（4）减少GABA的传输，这加剧了惊厥 症状（安定）。在建立涕灭威解毒剂发现系统的这项工作中，有两个目标。 第一个目标是确定涕灭威的麻痹活性，这种活性可以在人源化动物中容易地检测到 模型第二个目标是测量四种药物（阿托品、解磷定、地西泮）的解毒效果 和肉毒杆菌素）治疗涕灭威引起的瘫痪。当找到检测方案时，项目就成功了 能够检测解毒剂在降低涕灭威毒性方面的效果。在今后的工作中， 该系统可用作涕灭威解毒剂以及多种神经毒性药物的筛选平台， 威胁人类健康的物质。

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.