Epigenomic labeling of cells that drive drug abuse behavior

项目摘要 在努力为数千万慢性疼痛患者提供疼痛缓解的过程中，阿片类药物是最重要的药物之一。 在美国常用的处方药。因此，大部分人口面临着 阿片类药物的有害副作用，可能危及生命，包括成瘾和呼吸衰竭。 尽管有这些负面后果，但强迫性阿片类药物使用定义了阿片类药物使用障碍，这种情况是 每年造成近5万人死亡和800亿美元的医疗费用。因此，迫切需要 开发阿片类药物使用障碍的改进治疗方法。 治疗阿片类药物使用障碍的最大挑战之一是其慢性性质，患者经常复发 在长时间的戒毒之后脑梗死患者中脑核持续性表观基因组改变 阿片类药物使用障碍被认为有助于其慢性复发过程。它仍然具有挑战性， 然而，将这些知识转化为新的治疗方法是不可能的， 选择性地靶向参与药物寻求行为的表观基因组修饰的神经元， 在不相关的电路中的邻近细胞。 在这里，我们提出了一种创新的方法来标记细胞，这些细胞基于其独特的阿片类药物寻求行为。 表观基因组图谱为此，我们将首先以单细胞分辨率绘制染色质区域， 吗啡自身给药后，小鼠延髓核神经元选择性接近， 阿片类药物使用障碍的模型。这些基因组区域中的一些将作为功能基因调控元件 其在吗啡自身给药后激活基因表达（例如基因增强子）。识别这些 功能基因增强子，我们将产生一个腺相关病毒文库，其中每个推定的元件 促进独特条形码的表达。然后我们将使用单核RNA测序来快速筛选 该体内病毒文库用于选择性地驱动其条形码在细胞核中表达的元件 吗啡自我给药后表观基因组改变的神经元。最具选择性的 候选病毒将用于表达抑制性化学发生通道以控制吗啡寻求 行为 该提案的成功完成将建立一种全新的方法来选择性地标记，纯化， 和控制阿片类药物寻找行为的细胞。这种方法提供了许多优于当前的方法的优点。 最先进的技术，包括标记参与药物寻求行为的细胞的能力，而不需要 转基因小鼠或精确定时的条件刺激。通过使用进化上保守的基因调控 元件驱动病毒表达，这种方法也有可能转化为难治性 阿片类药物使用障碍

PROJECT SUMMARY In an effort to provide pain relief to tens of millions of patients with chronic pain, opioids are one of the most commonly prescribed medications in the United States. Large segments of the population are thus exposed to the detrimental side effects of opioids, which can be life threatening and include addiction and respiratory failure. Compulsive opioid use despite these negative consequences defines opioid use disorder, a condition that is responsible for nearly 50,000 deaths and $80 billion in medical costs annually. Thus, there is an urgent need for the development of improved treatments for opioid use disorder. One of the greatest challenges in treating opioid use disorder is its chronic nature with patients often relapsing after long periods of drug abstinence. Persistent epigenomic changes in the nucleus accumbens of patients with opioid use disorder are thought to contribute to its chronic, relapsing course. It has remained challenging, however, to translate this knowledge into novel therapeutic approaches because it has not been possible to selectively target the epigenomically-modified neurons involved in drug-seeking behavior without also affecting nearby cells in unrelated circuits. Here we present an innovative approach to label cells that drive opioid-seeking behavior based on their unique epigenomic profile. To do this, we will first map at single-cell resolution, the regions of chromatin that are selectively accessible in mouse nucleus accumbens neurons after morphine self-administration, an established model of opioid use disorder. Some of these genomic regions will act as functional gene regulatory elements that activate gene expression (e.g. gene enhancers) after morphine self-administration. To identify these functional gene enhancers, we will generate an adeno-associated viral library in which each putative element promotes the expression of a unique barcode. We will then use single-nucleus RNA-sequencing to rapidly screen this viral library in vivo for the elements that selectively drive expression of their barcode in the nucleus accumbens neurons that have been epigenomically altered by morphine self-administration. The most selective viral candidate will be used to express inhibitory chemogenetic channels for controlling morphine-seeking behavior. Successful completion of this proposal will establish a fundamentally new approach to selectively label, purify, and control cells that drive opioid-seeking behavior. This approach offers a number of advantages over current state-of-the-art technologies including the ability to label cells involved in drug-seeking behavior without need for transgenic mice or precisely timed conditioned stimuli. By using evolutionarily-conserved gene regulatory elements to drive viral expression, this approach also has the potential to translate to patients with refractory opioid use disorder.