Genetic Variation of Ultra-Potent Synthetic Opioid Sensitivity in Mice

小鼠超强合成阿片类药物敏感性的遗传变异

• 批准号: 10743432
• 负责人: Jason A Bubier
• 金额: $ 45.61万
• 依托单位: JACKSON LABORATORY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10743432
• 关键词: Acute; Address; Affect; African American population; Apnea; Automobile Driving; Biological; Blood; Blood gas; Brain Stem; Candidate Disease Gene; Carbon Dioxide; Cardiovascular system; Cations; Cause of Death; Cells; Central Nervous System; Cessation of life; Chest wall structure; Clustered Regularly Interspaced Short Palindromic Repeats; Coupled; Data; Development; Dose; Epidemic; Exhibits; Fentanyl; Foundations; Gene Expression; Genes; Genetic; Genetic Variation; Genetic study; Genotype; Goals; Human; Inbred Strains Mice; Individual Differences; Knock-out; Lethal Dose 50; Lung; Measures; Molecular; Monitor; Morphine; Moscow; Mouse Strains; Mus; Naloxone; Opioid; Opioid Analgesics; Opioid Antagonist; Opioid Receptor; Overdose; Oxygen; Pathway interactions; Patients; Peripheral Nervous System; Phenotype; Physiological; Plethysmography; Population; Recovery; Respiratory Mechanics; Signal Transduction; Street Drugs; Testing; Therapeutic; Tidal Volume; Time; Tissue-Specific Gene Expression; Toxic effect; Toxicokinetics; Variant; Ventilatory Depression; Withdrawal; Wooden Chest Syndrome; Work; addiction; aerosolized; analog; behavioral phenotyping; carfentanil; cell type; cohort; detection platform; differential expression; electric impedance; genome wide association study; hemodynamics; individual variation; innovation; insight; molecular phenotype; neural; novel; opioid overdose; overdose death; overdose risk; preBotzinger complex; prescription opioid; psychologic; respiratory; response; segregation; sex; single nucleus RNA-sequencing; synthetic opioid; targeted treatment; therapeutic development; translational impact; ventilation; weapons

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.

项目概要/摘要 超强合成阿片类药物 (UPS) 的武器化使得寻找新型逆转剂成为当务之急。 目前的阿片类药物反应剂纳洛酮对 UPS 阿片类药物并不有效，并且不能逆转其中一种 我们的长期目标是确定阿片类药物的生物学基础。 过量风险并促进发现逆转芬太尼致死性的安全有效药物。 目的是确定芬太尼毒性个体差异的分子机制 在目标 1 中，我们计划鉴定改变 Mcoln1 对基因的影响的基因和变异体。 急性 UPS 阿片类药物毒性是在一项关于过量风险的 GWAS 研究中发现的，并有初步数据支持。 Mcoln1 的基因敲除会导致吗啡或芬太尼更快地导致死亡。 在更敏感的遗传背景 NOD/ShiLtJ 上额外 CRISPR 敲除 Mcoln1 并进行比较 随着 C57BL/6 敲除，我们将确定针对 UPS 阿片类药物芬太尼的 LD50。 我们还将使用我们的压电呼吸抑制检测系统来研究这些菌株。 在另一个队列中，对三种剂量的芬太尼的反应的力学以及肺和胸壁阻抗。 小鼠将通过体积描记法进行测试，以获取呼吸指标，例如潮气量和分钟 通气期间我们还将采集动脉血来测量血气中的氧气和二氧化碳。 体积描记法监测该水平对芬太尼的反应最后，另一组天真的和 将解剖接受芬太尼治疗的小鼠的脑干，并对其前 Bötzinger 复合物进行鉴定和分析。 通过单核 RNA-Seq，比较细胞群体和差异基因表达 在目标 2 中，我们计划确定基因型、性别和治疗方法。 八种近交系小鼠品系中芬太尼诱导的不同毒性和致死性的机制。 品系，这是合作杂交和先进小鼠种群的基础 我们已确定远交小鼠的多样性含有大约 4500 万个 SNP。 与目标 1 一样，在目标 2 中，八种菌株的两性芬太尼的 LD50 > 150 倍。 小鼠表型队列检测与 UPS 阿片类药物相关的多种现象，包括阿片类药物 诱发呼吸抑制 (OIRD)、阿片类药物诱发持续性呼吸暂停 (OIPA)、木箱综合症 （WCS），上半身闭合/塌陷和心血管/血流动力学紊乱。 结合通过单核RNA测序识别脑干内的细胞群 bötzinger 区域在未经处理和芬太尼治疗的两性小鼠品系中存在差异。 定义这些人群的表达基因将帮助我们确定治疗开发的目标 与不同的芬太尼致死表型相关。