Summary We have recently discovered macrophage ED2/CD163 gene overexpression as a novel and safe pain therapeutic target in the local peripheral immune system in a major surgery rat model. We propose to develop a cell-directed gene therapy that would correct the underlying local immunological cause of pain resulting from inflammatory processes, specifically in sub-chronic postoperative pain or inflammatory conditions. Through unbiased genome-wide transcriptomic analyses in human primary macrophages we identified that ED2/CD163 gene induction modulates tumor necrosis factor alpha (TNFa) and interleukin (IL)-1 beta (IL-1b). CD163 overexpression using a clinically tested nanoparticle designed to target macrophages promoted a more rapid wound healing in 3D human organotypic skin tissues and prevented sub-chronic postoperative pain behaviors and reduced local TNFa and IL-1b in rats with skin-muscle incision and retraction (SMIR) surgery. We hypothesize that ED2/CD163 in macrophages is a safe target for the treatment of inflammatory pain with opioid sparing effects. We propose a multidimensional research plan including, 1) a sub-chronic surgical pain model, the SMIR surgery, and a knee inflammatory mode, the Complete Freund Adjuvant (CFA)-induced knee arthritis; 2) ED2/CD163 gain and loss of function using macrophage-directed nanotechnology; 3) novel, clinically relevant, and complex operant pain-related behaviors; 4) cellular/molecular, tissue, and transcriptomic outcomes for mechanistic target engagement; and 5) studies for ED2/CD163’s effects on opioid requirements. We will implement our plan through these specific aims: 1) Determine that macrophage specific ED2/CD163 gene induction effectively promotes resolution of inflammatory pain. A mannosilated polyethyleneimine nanoparticles (Man-PEI) designed to deliver nucleic acids specifically to macrophages will be used to conduct ED2/CD163 gain (overexpression) or loss (knock down) of function. We will assess classic behaviors (von Frey and weight bearing), and novel complex and clinically relevant functional activity and attention-related behaviors developed and validated by our team. 2) Define ED2/CD163 target engagement, i.e. ED2/CD163 as a signaling driver that dictates the dynamics of macrophage phenotype change and cellular reprogramming in inflammatory pain. TNFa and IL-1b will be measured as downstream target engagement. Also, we will use single-cell RNAseq (scRNAseq), cluster, and phenotype trajectory analysis to define how ED2/CD163 impacts gene expression programs in macrophages infiltrating the inflamed tissue. 3) Establish that macrophage ED2/CD163 gene induction results in opioid-sparing effects in surgical and inflammatory pain. We will construct dose responses of morphin in rats with SMIR or arthritis and ED2/CD163 overexpression to measure opioid-sparing effects. Our project will establish ED2/CD163 as a cell-directed gene therapy for postsurgical pain that will reduce opioid requiremen...