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.