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Using CRISPR tools to uncover the role of CREB-gene regulation in drug abuse

使用 CRISPR 工具揭示 CREB 基因调控在药物滥用中的作用

基本信息

批准号：
10057492
负责人：
Peter James Hamilton
金额：
$ 24.9万
依托单位：
VIRGINIA COMMONWEALTH UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-05-01 至 2023-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10057492
关键词：
Affect Animal Model Behavior Behavioral Binding Binding Proteins Bioinformatics Brain CRISPR/Cas technology Cells Chronic Clustered Regularly Interspaced Short Palindromic Repeats Cocaine Cocaine Abuse Cocaine Dependence Complex Consumption Control Locus Coupled Cyclic AMP-Responsive DNA-Binding Protein DNA DNA Binding DNA-Binding Proteins Data Development Disease Drug Addiction Drug Exposure Drug abuse Engineering Epigenetic Process Etiology Event Exposure to FDA approved Family Financial cost Gene Expression Regulation Genes Genetic Genetic Transcription Genomic Segment Genomics Goals Guide RNA Inherited Injections Knowledge Lead Mediating Mentors Mentorship Modification Molecular Morphine Morphine Abuse Mus Neurobiology Neurons Nucleus Accumbens Opiate Addiction Opioid Pathogenesis Pathogenicity Patients Pharmaceutical Preparations Phase Physiological Predisposition Process Property Proteins Public Health Regulator Genes Repression Research Personnel Research Proposals Rewards Role Self Administration Simplexvirus Speed Substance abuse problem Sum Syndrome System Techniques Technology Testing Tissues Training Transcription Alteration Transcriptional Regulation United States Viral Volition Zinc Fingers addiction brain reward regions cell type differential expression drug of abuse drug sensitivity genomic locus in vivo insight neuropsychiatric disorder neuropsychiatry novel novel therapeutics nuclease opioid abuse overexpression promoter psychostimulant recruit relating to nervous system societal costs stem stimulant abuse targeted treatment tool transcription factor transcriptome sequencing

项目摘要

PROJECT SUMMARY Substance abuse, including the abuse of psychostimulants and opiates, represents a major public health concern that exacts tremendous financial and societal costs. Abuse of these drugs remain recalcitrant conditions with limited FDA-approved treatments. Elucidation of the differential neural molecular mechanisms underlying psychostimulant versus opiate abuse is necessary to the development of novel, targeted therapies. There is a substantial body of evidence indicating that epigenetic and transcriptional mechanisms contribute to the pathogenesis of both psychostimulant and opiate abuse and dependence. Changes in the expression levels and the genomic localization of transcription factors have been identified in drug-exposed animal models. For example, repeated exposure to opiates or to psychostimulants elevate cyclic AMP-responsive element binding protein (CREB)-mediated transcription in the nucleus accumbens (NAc), a major brain reward region. Over-expression of CREB decreases the rewarding effects of psychostimulants and opiates, suggesting that CREB activity in this region can regulate the addictive properties of these drugs. However, manipulations of this kind cause epigenetic and transcriptional changes at hundreds of loci, limiting mechanistic insight critical for the development of targeted addiction therapies. Moreover, reward-related transcriptional gene regulatory mechanisms appear to be cell-type specific, with distinct roles for the NAc medium spiny neuron (MSN) subtypes. Therefore, to determine the causal contribution of cell-type specific, gene locus-targeted CREB recruitment in the differential pathogenesis of psychostimulant versus opiate abuse, I have engineered CRISPR technology as a vehicle for the conditional, locus-specific recruitment of CREB. I fused the nuclease- dead, RNA-guided, DNA-binding protein dCas9 to the active form of CREB (S133D), and I am able to direct CREB within targeted cell types to desired genomic regions to modulate transcription in vivo. Under the primary mentorship of Drs. Eric Nestler and Li Shen, I will receive training on in vivo, cell-targeted CRISPR delivery strategies, drug self-administration, and bioinformatic approaches. This training will allow me to use these highly specific manipulations to investigate a complete perspective on their differential contribution to psychostimulant versus opiate pathogenic volitional behaviors and transcriptional networks. I will validate and optimize my technology at the well-studied Fosb locus, allowing for meaningful interpretation of my data, and leverage the speed inherent to the CRISPR system to target a novel, bioinformatically predicted loci, Zfp189. In my independent phase, I plan to build upon my training to multiplex CREB binding to a novel drug self- administration identified gene network. This would establish the potential of using these physiologically- relevant manipulations to move beyond conventional monogenic manipulations in understanding complex neuropsychiatric syndromes. This training will aid in my transition into an independent investigator and yield novel insight on the differential causal pathogenic mechanisms of psychostimulant versus opiate abuse.

项目摘要药物滥用，包括滥用精神兴奋剂和鸦片制剂，是一个主要的公共卫生问题，这需要付出巨大的经济和社会代价。这些药物的滥用仍然很猖獗 FDA批准的治疗方法有限。阐明差异神经分子机制潜在的精神兴奋剂与阿片类药物滥用是必要的新的，有针对性的治疗的发展。有大量证据表明，表观遗传和转录机制有助于精神兴奋剂和阿片类药物滥用和依赖的发病机制。表达的变化已经在药物暴露动物模型中鉴定了转录因子的水平和基因组定位。例如，反复暴露于阿片类药物或精神兴奋剂，结合蛋白（CREB）介导的转录在丘脑核（NAc），一个主要的大脑奖励区。 CREB的过度表达降低了精神兴奋剂和阿片类药物的奖励作用，这表明，该区域的CREB活性可以调节这些药物的成瘾性。然而，操纵这类基因在数百个基因座上引起表观遗传和转录变化，限制了关键的机制洞察力。用于开发针对性成瘾治疗。此外，奖励相关的转录基因调控机制似乎是细胞类型特异性的，与NAc中棘神经元（MSN）的不同作用亚型因此，为了确定细胞类型特异性、基因位点靶向CREB的因果作用，招募精神兴奋剂与阿片类药物滥用的不同发病机制，我已经设计了 CRISPR技术作为CREB有条件、基因座特异性招募的工具。我融合了核酸酶- 死亡，RNA引导，DNA结合蛋白dCas 9的CREB（S133 D）的活性形式，我能够直接 CREB在靶向细胞类型内的靶向基因组区域以调节体内转录。下在Eric Nestler博士和Li Shen博士的主要指导下，我将接受体内细胞靶向CRISPR的培训递送策略、药物自我给药和生物信息学方法。这次训练会让我这些高度具体的操作，以调查其差异贡献的完整视角，精神兴奋剂与阿片类致病性意志行为和转录网络。我会验证，优化我在研究充分的Fosb位点的技术，允许对我的数据进行有意义的解释，利用CRISPR系统固有的速度来靶向新的生物信息学预测的基因座Zfp 189。在我的独立阶段，我计划建立在我的训练，以多重CREB结合到一个新的药物自我，政府确定的基因网络。这将建立使用这些生理上的潜力- 相关的操作，以超越传统的单基因操作，在理解复杂的神经精神综合征这项培训将有助于我转变为一名独立调查员，对精神兴奋剂与阿片类药物滥用的不同因果致病机制的新见解。