Epigenome Editing in Opioid Action

阿片类药物作用中的表观基因组编辑

• 批准号: 10268223
• 负责人: Mary Kay Lobo
• 金额: $ 19.31万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-30 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10268223
• 关键词: Abstinence; Animals; Attention; Behavior; Behavioral; Brain; Brain region; CRISPR interference; Cell Nucleus; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Cocaine; Data; Dependence; Discipline; Dopamine Receptor; Epidemic; Epigenetic Process; Fentanyl; Fiber Optics; Fluorescence; Gene Expression; Genes; Genetic Transcription; Goals; Guide RNA; Health; Implant; Injections; Intake; Lead; Light; Lighting; Luciferases; Mediating; Molecular; Motivation; Mus; Neurobiology; Neurons; Neurosciences; Nucleus Accumbens; Opioid; Overdose; Pathway Analysis; Pharmaceutical Preparations; Phase; Predisposition; Process; Quantitative Reverse Transcriptase PCR; RNA; Regulation; Relapse; Renilla Luciferases; Sorting - Cell Movement; Source; Specificity; Stress; Testing; Time; Water; Zinc Fingers; addiction; behavioral outcome; behavioral response; brain cell; brain reward regions; cell type; chromatin immunoprecipitation; combat; design; drug of abuse; epigenome; epigenome editing; genomic locus; histone modification; improved; in vivo; knock-down; opioid abuse; opioid exposure; opioid overdose; opioid use; opioid use disorder; optogenetics; overexpression; prevent; social stress; synthetic opioid; tool; transcription factor; transcriptome sequencing; translatome; vector

Project Summary The goal of this project is to develop CRISPR epigenome editing tools that achieve cell type specificity and in some cases temporal specificity to target opioid regulated molecules. Opioid use, dependence, and addiction have dramatically increased to epidemic proportions in recent years, leading to substantial financial and societal health burdens, as well as an increasing number of overdoses. The number of encounters with synthetic opioids, primarily fentanyl, has increased 300% since 2014, resulting in an increase of 72% in synthetic opioid overdoses. To combat this epidemic it is imperative that we understand the neurobiological underpinnings that lead to opioid use disorder. We must identify disrupted neuron subtypes in the brain in opioid use disorders and dysregulated molecules within these neurons that underlie cellular, circuit, and ultimately behavioral adaptations. We must then develop more effective tools to target endogenous gene transcription of molecules disrupted in opioid used disorder. To do this we performed RNA-seq in nucleus accumbens medium spiny neuron subtypes, those expressing D1 or D2, in fentanyl abstinent animals, during a time point in which they display stress vulnerable behavior. We identified key gene expression networks that are altered in MSN subtypes of fentanyl abstinent mice. In this proposal we will develop CRISPR epigenome editing tools, including temporal specific Optogenetic CRISPR tools, for selective cell subtype targeting of key fentanyl regulated genes in D1- MSNs and D2-MSNs. We will test these tools in Neuro2a cells and then in vivo in MSN subtypes (R21). Validated tools will be used in vivo to alter major hub genes, highly connected genes that regulate expression of other genes within a network. RNA-seq on MSN subtypes during fentanyl abstinence will determine if CRISPR tools are effective in targeting hub genes to alter their gene co- expression networks (R33). In parallel we will test Opto-CRISPR tools to disrupt hub genes during discreet stress susceptible and opioid intake and relapse time points (R33). Collectively, our studies have the ability to uncover unique opioid molecular mechanisms by targeting the epigenome at endogenous gene loci. Importantly our developed tools can be applied across all brain regions, cell subtypes, and neuroscience disciplines.

项目摘要 该项目的目标是开发CRISPR表观基因组编辑工具，实现细胞类型特异性 并且在某些情况下对靶阿片调节分子具有时间特异性。阿片类药物使用、依赖和 近年来，成瘾已急剧增加到流行病的比例，导致大量的 财政和社会健康负担，以及越来越多的过量用药。的数量 自2014年以来，与合成阿片类药物（主要是芬太尼）的接触增加了300%， 合成阿片类药物过量增加72%。为了防治这一流行病，我们必须 了解导致阿片类药物使用障碍的神经生物学基础。我们必须确定 阿片类药物使用障碍中大脑中神经元亚型的破坏和 这些神经元构成了细胞、电路和最终行为适应的基础。那么我们必须 开发更有效的工具来靶向阿片类药物中被破坏的分子的内源性基因转录 使用的障碍。为了做到这一点，我们进行了RNA-seq中的神经元 亚型，表达D1或D2的那些，在芬太尼戒断动物中，在一个时间点， 表现出易受压力影响的行为我们确定了关键的基因表达网络， 芬太尼戒断小鼠的MSN亚型。 在这项提案中，我们将开发CRISPR表观基因组编辑工具，包括时间特异性 光遗传学CRISPR工具，用于选择性细胞亚型靶向D1中的关键芬太尼调控基因- MSN和D2-MSN。我们将在Neuro2a细胞中测试这些工具，然后在MSN亚型中进行体内测试 （R21）。经过验证的工具将在体内用于改变主要枢纽基因，这些基因高度连接， 调节网络中其他基因的表达。芬太尼给药期间MSN亚型的RNA-seq 禁欲将决定CRISPR工具是否有效地靶向枢纽基因，以改变它们的基因协同作用。 表达网络（R33）。与此同时，我们将测试Opto-CRISPR工具，以破坏枢纽基因， 离散的压力敏感和阿片类药物摄入和复发时间点（R33）。总的来说，我们的研究 有能力通过靶向表观基因组， 内源基因座重要的是，我们开发的工具可以应用于所有的大脑区域，细胞 亚型和神经科学学科。