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

总结 我们最近发现巨噬细胞ED 2/CD 163基因过表达作为一种新的和安全的疼痛 在大手术大鼠模型中局部外周免疫系统中的治疗靶点。我们建议发展一个 细胞导向的基因治疗，将纠正潜在的局部免疫性疼痛的原因， 炎症过程，特别是在亚慢性术后疼痛或炎症状况。通过 在人原代巨噬细胞中进行的无偏全基因组转录组分析中，我们确定ED 2/CD 163 基因诱导调节肿瘤坏死因子α（TNF α）和白细胞介素（IL）-1 β（IL-1b）。CD163 使用临床测试的靶向巨噬细胞的纳米颗粒的过表达促进了更快的 3D人体器官型皮肤组织中的伤口愈合并预防亚慢性术后疼痛行为 并减少皮肤肌肉切开牵开术（SMIR）大鼠局部TNF α和IL-1b。我们 假设巨噬细胞中ED 2/CD 163是治疗炎性疼痛的安全靶点， 阿片类药物的节省作用。我们提出了一个多维的研究计划，包括：1）亚慢性手术疼痛 模型，SMIR手术，和膝关节炎症模型，完全弗氏佐剂（CFA）诱导的膝关节 关节炎; 2）使用巨噬细胞定向纳米技术的ED 2/CD 163功能获得和丧失; 3）新的，临床上 相关和复杂的操作性疼痛相关行为; 4）细胞/分子、组织和转录组学结果 5）ED 2/CD 163对阿片类药物需求的影响的研究。我们将 通过以下具体目标实施我们的计划：1）确定巨噬细胞特异性ED 2/CD 163基因 诱导有效地促进炎性疼痛的消退。一种甘露糖化聚乙烯亚胺， 设计用于将核酸特异性递送至巨噬细胞的纳米颗粒（Man-PEI）将用于进行 ED 2/CD 163功能获得（过表达）或丧失（敲低）。我们将评估典型的行为（冯弗雷 和负重），以及新的复杂的和临床相关的功能活动和注意力相关的行为 由我们的团队开发和验证。2)定义ED 2/CD 163目标接合，即ED 2/CD 163作为 决定巨噬细胞表型变化和细胞重编程动力学的信号驱动因子 炎症性疼痛。将测量TNFa和IL-1b作为下游靶点接合。此外，我们将使用 单细胞RNA测序（scRNA测序）、聚类和表型轨迹分析，以定义ED 2/CD 163的影响方式 浸润发炎组织的巨噬细胞中的基因表达程序。3)建立巨噬细胞 ED 2/CD 163基因诱导导致手术和炎性疼痛中的阿片样物质节省效应我们将 构建吗啡在SMIR或关节炎和ED 2/CD 163过表达大鼠中的剂量反应，以测量 阿片类药物的作用我们的项目将建立ED 2/CD 163作为手术后疼痛的细胞定向基因治疗 这将减少阿片类药物的需求，并揭示它如何影响免疫反应和炎症疼痛 复苏我们的多学科团队具有独特的能力来成功完成这些研究。

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.