Cell-directed gene therapy for pain recovery after surgery and inflammation

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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 requirements and disinter how it influences immune responses and inflammatory pain recovery. Our multidisciplinary team is uniquely equipped to successfully complete these studies.