Project Summary Chronic pain affects over 25 million adults in the United States and is a major cause of disability. Currently available pain treatments such as opioids are often ineffective and associated with unacceptable side effects including respiratory depression and addiction. A major goal for new pain therapeutics is to inhibit the sensory neurons which transmit pain signals (nociceptors) selectively without affecting other neurons involved in innocuous sensation or the central nervous system. However, nociceptor-specific therapeutic approaches remain in their infancy. Viral-based gene therapy offers several attractive advantages in treating refractory pain, as viruses can be engineered to deliver a wide range of molecules, can be administered locally or systemically, and have been recently approved by the Food and Drug Administration for a number of indications. However, nociceptor-specific viral tools do not presently exist in large part due to the extraordinary heterogeneity of sensory neurons that has made it difficult to identify molecular features that are unique to these cells. Recent advances in single-cell genomics have enabled us to generate a cell atlas that describes the genes that are selectively expressed in mouse and human nociceptors. This proposal aims to uncover the endogenous gene regulatory elements that mediate nociceptor-specific gene expression patterns and engineer these elements into barcoded adeno-associated viral libraries. To accomplish this, we propose the following specific aims: 1) Mapping nociceptor-specific gene regulatory elements in mouse and human and 2) Generation of a nociceptor-specific AAV toolkit. The nociceptor-specific viruses we develop in this proposal will be immediately useful to the scientific community for accessing nociceptors in wild-type mice and likely other species. In addition, because we will prioritize gene regulatory elements that are conserved between mouse and human nociceptors, we are optimistic that the viruses we screen in mice will also drive nociceptor-specific gene expression in humans. These next generation nociceptor-specific gene therapies would be ideally suited for treating certain refractory pain disorders because they can be administered locally to neuropathic sensory ganglia through standard outpatient procedures and can be engineered to drive expression of ion channels that silence nociceptor activity only in the presence of a specific drug (chemogenetics). This opioid-free approach could have significant translational applications for patients with refractory chronic pain who often have no other options for relief.