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

摘要 我们最近发现巨噬细胞ED2/CD163基因的过度表达是一种新的安全的疼痛 治疗靶点在大手术大鼠模型的局部外周免疫系统。我们建议开发一种 细胞导向的基因治疗将纠正引起疼痛的潜在局部免疫原因 炎症过程，特别是在亚慢性术后疼痛或炎症条件下。穿过 人原代巨噬细胞全基因组转录无偏分析我们鉴定了ED2/CD163 基因诱导调节肿瘤坏死因子α(TNFa)和白介素1β(IL-1b)。CD163 使用经过临床测试的针对巨噬细胞的纳米颗粒促进过度表达更快 3D人体器官型皮肤组织的伤口愈合和预防亚慢性术后疼痛行为 降低皮肤肌肉切开牵引术(SMIR)大鼠局部肿瘤坏死因子α(TNFa)和白介素1β(IL-1b)水平。我们 假设巨噬细胞中的ED2/CD163是治疗炎性疼痛的安全靶点 阿片类药物的节制作用。我们提出了一个多维度的研究计划，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)利用巨噬细胞导向的纳米技术获得和丧失ED2/CD163的功能；3)新的临床应用 相关和复杂的可操作性疼痛相关行为；4)细胞/分子、组织和转录结果 5)ED2/CD163‘S对阿片类药物需求的影响研究。我们会 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ED2/CD163基因 诱导能有效地促进炎性疼痛的消退。甘露硅化聚乙烯亚胺 专为巨噬细胞运送核酸的纳米粒子(Man-PEI)将被用于 ED2/CD163功能增强(过度表达)或丧失(下调)。我们将评估经典行为(冯·弗雷 和负重)，以及新的复杂和临床相关的功能活动和注意相关行为 由我们的团队开发和验证。2)定义ED2/CD163目标接洽，即ED2/CD163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素 在炎性疼痛中。TNFa和IL-1b将作为下游目标参与来衡量。此外，我们还将使用 单细胞RNAseq(ScRNAseq)、集群和表型轨迹分析，以定义ED2/CD163如何影响 巨噬细胞中的基因表达程序渗入炎症组织。3)建立巨噬细胞 ED2/CD163基因诱导在手术和炎症性疼痛中产生阿片类药物节省效应。我们会 构建吗啡在Smir或关节炎大鼠体内的剂量效应及ED2/CD163过度表达的测量 阿片类药物的节制作用。我们的项目将建立ED2/CD163作为一种针对术后疼痛的细胞导向基因疗法 这将减少阿片类药物的需求，并揭示它是如何影响免疫反应和炎性疼痛的 恢复。我们的多学科团队具备成功完成这些研究的独特条件。

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.