Project Abstract – Project 1 Chronic pain afflicts over 100 million Americans and is a leading reason for seeking medical treatment. Few new therapies have been delivered in the past decades, resulting in overuse of opioids, despite their potential for abuse and limited efficacy in many chronic pain conditions. One glaring concern is the failure of studies using preclinical models to yield agents that are effective pain therapeutics. A key barrier in addressing this problem is the knowledge gap between our understanding of the human peripheral nervous system and the studies performed in rodent preclinical pain models. In this project, we propose to identify additional genes involved in human pain pathways via analysis of the genomes of a cohort of patients with idiopathic painful neuropathy. While nociceptive neurons are the components most critical for peripheral pain transmission, other cells in peripheral nerves—glia and immune cells, particularly macrophages—are important regulators of pain responses in these neurons. Additional analysis is required to understand how interactions among these cellular nerve components, and with sensory axons, contribute to pain. We therefore propose to use single-nuclei RNAseq strategies that we recently utilized to produce a mouse peripheral nerve atlas to create a multi-layered, comprehensive atlas of the expression profiles of human peripheral nerve cellular components. In this proposal, we outline a series of experiments to examine gene expression in cells of human peripheral nerve and to identify novel gene variants associated with idiopathic painful neuropathy. Whole genome sequencing will be performed on samples from a large cohort of well-characterized patients with painful neuropathy. Gene variants will be associated with phenotypic information to identify new genes involved in the pain pathway. Using multi-omic technologies, including snRNA-seq and snATAC-seq as well as spatial transcriptomics and multiplex proteomics, to study nerves from our donor tissue core, we will devise a comprehensive, unbiased single-nuclei atlas of gene expression and cellular composition of human peripheral nerves. Parallel studies will be performed to determine the cellular components and gene expression patterns of surgically resected painful neuromas. By comparing the neuroma transcriptomes to the atlas derived from normal nerve datasets, we plan to identify cell type-specific determinants of painful neuromas. We expect these rich and complementary datasets to provide a foundation for molecular and cellular analysis of sensory nerve dysfunction in pain syndromes that will provide insights into treatment options for pain.