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变化和疼痛之间潜在联系的理解是不完整的， 这直接限制了我们开发靶向和有效的治疗策略来治疗椎间盘源性疼痛的能力。 此外，我们能够同时独立地针对退行性变化和疼痛 是无法实现的。IVD变性和疼痛与促炎性环境（即TNF-α、IL-1β、IL-6、IL-10、IL-11、IL-12、IL-14、IL-15、IL-16、IL-17、IL-18、IL-19、 IFN-γ），可导致IVD内的椎间盘退行性变以及神经支配的伤害性神经元的致敏 IVD同时针对这两种不同的结果（ECM变化和神经元致敏） 将提供新的治疗策略，既可以治疗疼痛，又可以预防疾病进展。我们 将利用CRISPR表观基因组编辑提供TNF-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传导的途径， 退行性椎间盘疾病进展。在目的1中，我们将研究炎症受体的调节， 背根神经节与CRISPR表观基因组编辑，以改善退行性椎间盘疾病的疼痛。我们将 研究TNFR 1，IL 1 R1，IL 6ST和TNFR 1/IL 1 R1/IL 6ST同时靶向伤害性刺激， 体外和体内的神经元。使用具有预防相关性的人类退行性IVD组织模型 伤害感受神经元敏化，我们研究热（目的1A）和机械（目的1B）敏化 这些研究表明，CRISPR是一种重要的生物学途径，我们有能力在体外使用CRISPR表观基因组编辑来调节它们。(Aim 1C）我们将交付 在疼痛的啮齿动物中，在两个递送时间点（0和4周）将慢病毒表观基因组编辑载体递送至DRG 腰椎环形穿刺模型。在急性和慢性时间点，疼痛相关的机械测量 异常性疼痛和热痛觉过敏将在清醒的动物中获得，在DRG和背角钙之前 显像这些结果将提供关于炎症反应中多余炎症信号的关键信息。 退行性椎间盘疾病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 研究IVD中两个递送时间点（0周和4周）的炎症受体调节， CRISPR表观基因组编辑可减缓退行性椎间盘疾病的进展来调查我们的能力 调节退行性疾病的进展，并研究急性和慢性炎症中TNF- α、IL-1 β和IL-6信号传导， 慢性疾病进展，我们将同时靶向TNFR 1、IL 1 R1、IL 6ST和TNFR 1/IL 1 R1/IL 6ST， 将慢病毒表观基因组编辑载体递送至啮齿动物环状穿刺模型中的IVD。我们将 研究椎间盘高度、ECM结构和成分变化以及炎症，以评估 多余的TNF-α，IL-1β和IL-6在椎间盘退变中的作用，并了解我们预防椎间盘退变的能力。 退行性椎间盘疾病进展。在急性和慢性时间点， 在DRG和背角之前，将在清醒动物中获得机械异常性疼痛/热痛觉过敏 钙成像，以确定同时改变IVD变性进展和伤害感受的能力。