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.