Sequence-specific CRISPR mediated inflammatory cytokine receptor modulation for the treatment of inflammatory intervertebral disc pathology

序列特异性 CRISPR 介导的炎性细胞因子受体调节用于治疗炎性椎间盘病理

• 批准号: 10454149
• 负责人: Robert D. Bowles
• 金额: $ 33.21万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10454149
• 关键词: Acute; Acute Disease; Animals; Back Pain; Blocking Antibodies; Calcium; Cells; Chronic; Chronic Disease; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Cytokine Receptors; Cytokine Signaling; Data; Degenerative Disorder; Development; Disease; Disease Progression; Extracellular Matrix; Genes; Height; Human; IL6 Signaling Pathway; IL6ST gene; Image; In Vitro; Individual; Inflammation; Inflammatory; Inflammatory Response; Interferon Type II; Interleukin-1; Interleukin-1 Receptors; Interleukin-1 beta; Interleukin-6; Intervertebral disc structure; Lead; Lentivirus; Link; Measures; Mechanics; Mediating; Modeling; Molecular; Motivation; Neurons; Nociception; Nociceptors; Outcome; Pain; Pain management; Pathologic; Pathology; Pathway interactions; Puncture procedure; Regulation; Research; Rodent; Role; Signal Transduction; Spinal Ganglia; Structure; System; TNF gene; TNFRSF1A gene; Technology; Testing; Therapeutic; Thermal Hyperalgesias; Time; Tissue Model; Tissues; awake; base; cytokine; discogenic pain; dorsal horn; epigenome; epigenome editing; improved; in vivo; inflammatory milieu; intervertebral disk degeneration; mechanical allodynia; novel; pain signal; prevent; receptor; receptor expression; response; therapeutically effective; treatment strategy; vector

Our understanding of the underlying links between degenerative changes in the disc and pain is incomplete, which directly limits our ability to develop targeted and effective therapeutic strategies to treat discogenic pain. Additionally, our ability to both target the degenerative changes and the pain simultaneously and independently is unrealized. IVD degeneration and pain are linked by a pro-inflammatory environment (i.e. TNF-α, Il-1β, Il-6, IFN-γ) that can lead to disc degeneration within the IVD and sensitization of nociceptive neurons that innervate the IVD. Targeting these two distinct outcomes (ECM changes and neuronal sensitization) simultaneously would provide novel treatment strategies that could both treat the pain and prevent disease progression. We will utilize CRISPR epigenome editing to provide simultaneous targeting of the TNF-α, IL-1β, and IL-6 signaling pathways through receptor modulation both in the DRG and directly in the IVD to alter pain signaling and degenerative disc disease progression. In Aim 1, we will investigate regulation of inflammatory receptors in the dorsal root ganglia with CRISPR epigenome editing to modify pain in degenerative disc disease. We will investigate the targeting of TNFR1, IL1R1, IL6ST, and TNFR1/IL1R1/IL6ST simultaneously in nociceptive neurons in vitro and in vivo. Using a translationally relevant human degenerative IVD tissue model of nociceptive neuron sensitization, we investigate thermal (Aim 1A) and mechanical (Aim 1B) sensitization pathways and our ability to modulate them using CRISPR epigenome editing in vitro. (Aim 1C) We will deliver lentiviral epigenome editing vectors to the DRG at two delivery time points (0 and 4 weeks) in a painful rodent lumbar annular puncture model. At both acute and chronic time points, pain related measures of mechanical allodynia and thermal hyperalgesia will be obtained in awake animals, prior to DRG and dorsal horn calcium imaging. These outcomes will provide critical information on redundant inflammatory signaling in the development of pain in degenerative disc disease and our ability to modulate it in the DRG. In Aim 2, we will investigate the regulation of inflammatory receptors at two delivery time points (0 and 4 weeks) in the IVD with CRISPR epigenome editing to slow progression of degenerative disc disease. To investigate our ability to modulate degenerative disease progression and investigate TNF- α, IL-1 β and IL-6 signaling in acute and chronic disease progression, we will target TNFR1, IL1R1, IL6ST, and TNFR1/IL1R1/IL6ST simultaneously by delivering lentiviral epigenome editing vectors to the IVD in a rodent annular puncture model. We will investigate disc height, changes in ECM structure and composition, and inflammation to assess a role for redundant TNF-alpha, IL-1β, and IL-6 in disc degeneration and to understand our ability to prevent degenerative disc disease progression. At both acute and chronic time points, pain related measures of mechanical allodynia/thermal hyperalgesia will be obtained in awake animals, prior to DRG and dorsal horn calcium imaging to determine ability to simultaneously modify IVD degeneration progression and nociception.

我们对椎间盘退行性改变和疼痛之间的潜在联系的理解是不完整的， 这直接限制了我们开发有针对性和有效的治疗策略来治疗椎间盘源性疼痛的能力。 此外，我们同时和独立地针对退行性变化和疼痛的能力 是没有实现的。血管内皮细胞变性和疼痛由促炎环境(即肿瘤坏死因子-α、IL-1β、IL-6、 干扰素-γ)，可导致下丘脑内盘退变和支配伤害性感受神经元的敏化 移民局。同时针对这两种不同的结果(细胞外基质变化和神经元敏化) 将提供新的治疗策略，既可以治疗疼痛，又可以防止疾病进展。我们 将利用CRISPR表观基因组编辑来提供对肿瘤坏死因子-α、IL-1β和IL-6信号的同时靶向 通过DRG和直接在IVD中的受体调节来改变疼痛信号和 退行性间盘疾病进展。在目标1中，我们将研究炎症受体在血管内皮细胞中的调节 背根神经节与CRISPR表观基因组编辑，以缓解退行性腰椎间盘疾病的疼痛。我们会 同时靶向TNFR1、IL1R1、IL6ST和TNFR1/IL1R1/IL6ST在伤害性反应中的作用 体外和体内的神经元。使用翻译相关的人类退行性IVD组织模型 伤害性神经元敏化，我们研究了热敏化(目标1A)和机械敏化(目标1B) 途径以及我们使用CRISPR表观基因组编辑在体外调节它们的能力。(目标1C)我们将交付 在疼痛的啮齿动物的两个交付时间点(0和4周)将慢病毒表观基因组编辑载体转移到DRG 腰椎环状穿刺法。在急性和慢性时间点，与疼痛相关的机械性测量 在DRG和背角钙离子之前，清醒动物会出现痛觉过敏和热痛觉过敏 成像。这些结果将提供有关冗余炎症信号的关键信息。 退行性间盘疾病中疼痛的发展以及我们在DRG中调节疼痛的能力。在目标2中，我们将 观察两个分娩时间点(0周和4周)IVD时炎性受体的调节 CRISPR表观基因组编辑以减缓退行性间盘疾病的进展。为了调查我们是否有能力 调节退行性疾病进展并研究急性和慢性前列腺癌患者肿瘤坏死因子-α、IL-1β和IL-6信号转导途径 慢性疾病进展，我们将同时针对TNFR1、IL1R1、IL6ST和TNFR1/IL1R1/IL6ST，通过 在啮齿动物环状穿刺法模型中向IVD递送慢病毒表观基因组编辑载体。我们会 调查椎间盘高度、ECM结构和成分的变化以及炎症，以评估其作用 冗余的肿瘤坏死因子-α、白介素1β和白介素6在椎间盘退变中的作用 退行性间盘疾病进展。在急性和慢性时间点，疼痛相关的测量 在DRG和背角之前，清醒的动物会出现机械性超敏/热痛觉过敏 钙成像以确定同时改变IVD退变进程和伤害性感觉的能力。