Genetic Variation of Ultra-Potent Synthetic Opioid Sensitivity in Mice

项目总结/摘要 超强效合成阿片类药物（UPS）的武器化使得寻找新型逆转剂成为当务之急。的 目前的阿片类药物反应剂纳洛酮对UPS阿片类药物不那么有效，并且不能逆转 它的已知症状木胸综合症我们的长期目标是确定阿片类药物的生物学基础 过量的风险，并促进发现安全有效的药物，扭转芬太尼的致命性。一个关键 目的是确定芬太尼毒性个体差异的分子机制， 基因多样的老鼠在目标1中，我们计划鉴定修改Mcoln 1对 急性UPS阿片类药物毒性。Mcoln 1是在一项关于药物过量风险的GWAS研究中发现的，初步数据支持 Mcoln 1基因敲除导致吗啡或芬太尼更快死亡。我们将创建一个 在更敏感的遗传背景NOD/ShiLtJ上敲除Mcoln 1的额外CRISPR，并将其与 C57 BL/6基因敲除后对UPS阿片类芬太尼的反应LD 50将测定为 这些菌株使用我们的压电呼吸抑制检测系统。我们还将研究呼吸系统 力学和肺和胸壁阻抗响应于三个剂量的芬太尼。在另一个队列中， 将通过体积描记法测试小鼠，以获得呼吸指标，如潮气量和分钟呼吸量。 通风.我们还将收集动脉血，以测量血液中的氧气和二氧化碳气体， 体积描记术以监测对该水平芬太尼的反应。最后，另一群天真的， 解剖芬太尼处理的小鼠的脑干。前波青格复合体将被识别和分析 通过单核RNA-Seq，比较细胞群体和不同的基因表达， 基因型、性别和治疗。在aim 2中，我们计划识别生理的，神经的和分子的 在8个近交系小鼠品系中，芬太尼诱导的毒性和致死率的变化机制。这八 品系，这是协作交叉的先进小鼠种群的基础， 远交系小鼠含有大约4500万个SNP，在它们之间分离。我们已经确定 芬太尼的LD 50在8种菌株的两种性别之间变化> 150倍。在目标1中，我们将 表型队列的小鼠，以检测与UPS阿片类药物，包括阿片- 诱导性呼吸抑制（OIRD）、阿片类药物诱导的持续性呼吸暂停（OIPA）、木胸综合征 (WCS)上呼吸道闭合/塌陷和心血管/血流动力学紊乱。这种表型将是 再加上识别细胞群体，通过单核RNA-Seq，在脑干前， bötzinger区域，其在两种性别小鼠的未处理和芬太尼处理品系之间变化。的差异 定义这些人群的表达基因将帮助我们确定治疗发展的目标 与不同的芬太尼致死性表型相关。

PROJECT SUMMARY/ABSTRACT The weaponization of Ultrapotent synthetic opioids (UPS) has made finding a novel reversal agent a priority. The current opioid response agent, Naloxone, is not as effective against UPS opioids and does not reverse one of its known effects, wooden chest syndrome. Our long-term goal is to define the biological basis of opioid overdose risk and promote the discovery of safe and effective agents that reverse fentanyl lethality. A key objective is determining the molecular mechanisms underlying individual variability to fentanyl toxicity using genetically diverse mice. In aim 1 we plan to identify genes and variants that modify the influence of Mcoln1 on acute UPS opioid toxicity. Mcoln1 was identified in a GWAS study of overdose risk, and preliminary data support a genetic knockout of Mcoln1 resulting in death more rapidly from morphine or fentanyl. We will create an additional CRISPR knockout of Mcoln1 on a more sensitive genetic background, NOD/ShiLtJ and compare it with the C57BL/6 knockout we have for response to the UPS opioid fentanyl. The LD50 will be determined for these strains using our piezoelectric respiratory depression detection system. We will also study respiratory mechanics and pulmonary and chest wall impedance in response to three doses of fentanyl. In another cohort of mice, they will be tested by plethysmography to acquire respiratory metrics such as tidal volume and minute ventilation. We will also collect arterial blood to measure blood gases of oxygen and carbon dioxide during the plethysmography session to monitor the response to fentanyl at that level. Finally, another cohort of naïve and fentanyl-treated mice will be dissected for brain stems. The pre-bötzinger complex will be identified and analyzed by single-nucleus RNA-Seq, comparing the cellular populations and differential gene expression across genotypes, sexes and treatments. In aim 2 we plan to identify the physiological, neural, and molecular mechanisms of variable fentanyl-induced toxicity and lethality among eight inbred mouse strains. These eight strains, which served as the foundation for the advanced mouse populations of the Collaborative Cross and Diversity Outbred mice, contain approximately 45 million SNPs segregating between them. We have determined that the LD50 for fentanyl varies > 150-fold across both sexes of the eight strains. As in aim 1, in aim 2 we will phenotype cohorts of mice to detect the diverse phenomena associated with UPS opioids, including Opioid- Induced Respiratory Depression (OIRD), Opioid-Induced Persistent Apnea (OIPA), Wooden Chest Syndrome (WCS), closure/collapse of the upper and cardiovascular/hemodynamic disturbances. This phenotyping will be coupled to identifying the cellular populations, through single nucleus RNA-Seq, within the brainstem pre- bötzinger region that varies across naïve and fentanyl-treated strains of both sexes of mice. The differentially expressed genes that define these populations will help us identify targets for therapeutic development associated with the different fentanyl lethality phenotypes.