Epigenomic labeling of cells that drive drug abuse behavior

项目概要 为了缓解数千万慢性疼痛患者的疼痛，阿片类药物是最有效的药物之一 在美国常用的处方药。因此，很大一部分人口暴露于 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，但强迫性使用阿片类药物定义了阿片类药物使用障碍，这是一种病症 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 开发针对阿片类药物使用障碍的改进疗法。 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 长期戒毒后。患者伏隔核持续的表观基因组变化 阿片类药物使用障碍被认为是导致其慢性、复发的原因。仍然充满挑战， 然而，要将这些知识转化为新的治疗方法，因为还不可能 选择性地靶向参与药物寻求行为的表观基因组修饰神经元，而不影响 不相关电路中的附近单元。 在这里，我们提出了一种创新方法来标记细胞，这些细胞根据其独特的特性驱动阿片类药物寻求行为 表观基因组概况。为此，我们首先以单细胞分辨率绘制染色质区域 吗啡自我给药后，可选择性地进入小鼠伏隔核神经元，这是一种已建立的方法 阿片类药物使用障碍模型。其中一些基因组区域将充当功能性基因调控元件 吗啡自我给药后激活基因表达（例如基因增强子）。为了识别这些 功能基因增强子，我们将生成一个腺相关病毒文库，其中每个假定的元素 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 该病毒体内文库包含选择性驱动其条形码在细胞核中表达的元件 伏隔神经元已通过吗啡自我给药而发生表观基因组改变。最有选择性的 候选病毒将用于表达抑制性化学遗传学通道，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、 并控制驱动阿片类药物寻求行为的细胞。与当前的方法相比，这种方法具有许多优点 最先进的技术，包括无需人工干预即可标记参与药物寻求行为的细胞的能力 转基因小鼠或精确定时的条件刺激。通过使用进化保守的基因调控 驱动病毒表达的元件，这种方法也有可能转化为难治性患者 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.