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

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 - andquot;Botoxandquot;, (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.Narrative Funding for the project will create a platform for discovery of antidotes to nerve agents. A humanized C. elegans animal model is used to achieve a better translation of study results to human use. Agents to prevent aldicarb toxicity is the focus of this preliminary study.