Sequence-Specific CRISPR Mediated Inflammatory Cytokine Receptor Modulation for the Treatment of Inflammatory Intervertebral Disc Pathology

 描述(申请人提供)：椎间盘的病理(IVD)和相关的背部疼痛是一个主要的医疗保健问题，在疾病负担(伤残调整寿命年)中排名第三。炎症和相关的促炎细胞因子(如肿瘤坏死因子-α、白细胞介素1？、白细胞介素6、白细胞介素8)被认为是许多与腰痛相关的事件和过程中的活性成分，包括腰椎间盘退变、IVD突出以及神经根病和退变的伤害性神经元的敏化。因此，这种细胞因子网络成为下腰痛治疗的主要靶点。由于这些细胞因子的显著冗余，需要能够在IVD中同时靶向多种炎性细胞因子的治疗方法。使用CRISPR系统抑制和激活多个基因的策略将提供一个强大的工具来对抗IVD病理中活跃的冗余促炎细胞因子网络。CRISPR调节方法能够在单一系统中提供高特异性、持续效应和多重能力。这项拟议的计划旨在开发基于CRISPR的退变间盘炎症信号调节的新用途，并测试其作为治疗策略的潜力。在目标1中，我们将验证CRISPR基因调控系统可以在功能上调节退变椎间盘中发现的细胞类型的炎症反应的假设。在目标1A中，我们将建立基于CRISPR的序列特异性下调(CRISPRi)，以调控参与椎间盘退变的关键炎性细胞因子受体的基因表达。将开发针对肿瘤坏死因子-α、IL-1β和IL-6受体的慢病毒载体(即TNFR1i、IL1R1i、IL6Ri、IL6STi)，并筛选其对调节人NP细胞、人背根神经节神经元和人巨噬细胞受体提呈的有效性。在目标1B中，我们将开发基于CRISPR的抗炎受体基因表达的序列特异性上调(CRISPRa)。针对IL1R2和TNFR2的表达DCAS-VP64和引导RNA的慢病毒载体将被开发并评估其在NP细胞、人背根神经节神经元和人巨噬细胞中的有效性。在目标1A和1B中，基于CRISPR的受体调节后的细胞对炎性细胞因子暴露的反应将使用定量聚合酶链式反应和核因子-κB报告程序来测量。在目标2中，我们将验证CRISPR基因调控系统可以调节炎症对IVD细胞的影响，并在炎症环境中促进IVD组织功能发育的假设。我们将使用基于CRISPR的炎性受体的序列特异性多重调节来对抗IVD细胞和组织中炎症的有害退化效应。炎症条件下的功能性血管内皮细胞发育和细胞反应将通过定量聚合酶链式反应(一种核因子-κB发光报告)、机械测试以及胶原和蛋白多糖的组织学/生物化学来评估。

 DESCRIPTION (provided by applicant): Pathology of the intervertebral disc (IVD) and associated back pain is a major healthcare concern, which ranks third in disease burden (disability adjusted life years). Inflammation and related pro-inflammatory cytokines (e.g. TNF-a, Il-1ß, Il-6, Il-8) have been implicated as active components in many events and processes associated with back pain, including disc degeneration, IVD herniation, and the sensitization of nociceptive neurons both in radiculopathy and in the degenerative disc. As a result, this network of cytokines is a primary target for low back pain therapeutics. Due to significant redundancy in these cytokines, there is a need for therapeutics that are capable of targeting multiple inflammatory cytokines simultaneously in the IVD. Strategies to inhibit and activate multiple genes using the CRISPR system will provide a powerful tool to antagonize the redundant pro-inflammatory cytokine network active in IVD pathology. The CRISPR regulation method is capable of providing high specificity, sustained effects, and multiplex capabilities in a single system. The proposed plan has been designed to develop the novel utility of CRISPR based regulation of inflammatory signaling in the degenerative disc, and test its potential as a therapeutic strategy. In aim 1, we will test the hypothesis that CRISPR gene regulation systems can functionally modulate inflammatory response in cell types found in the degenerative disc. In Aim 1A, we will develop CRISPR-based sequence-specific down regulation (CRISPRi) of gene expression of key inflammatory cytokine receptors involved in disc degeneration. Lentiviral vectors expressing dCAS-KRAB and guide RNAs targeting receptors for TNF-α, Il-1β and Il-6 will be developed (i.e. TNFR1i, IL1R1i, Il6Ri, Il6STi) and screened for efficacy in regulating receptor presentation in human NP cells, human DRG neurons, and human macrophages. In Aim 1B, we will develop CRISPR-based sequence-specific up regulation (CRISPRa) of gene expression of anti-inflammatory receptors. Lentiviral vectors expressing dCAS-VP64 and guide RNAs targeting Il1R2 and TNFR2 will be developed and evaluated for efficacy in NP cells, human DRG neurons, and human macrophages. In both aim 1A and 1B, the cells response to inflammatory cytokine exposure after CRISPR-based receptor modulation will be measured using qPCR and an NF-κB reporter. In Aim 2, we will test the hypothesis that CRISPR gene regulation systems can modulate the effects of inflammation on IVD cells and promote functional IVD tissue development in an inflammatory environment. We will use CRISPR-based sequence-specific multiplex regulation of inflammatory receptors to antagonize deleterious degenerative effects of inflammation in IVD cells and tissue. Functional IVD tissue development and cell response under inflammatory conditions will be assessed via qPCR, an NF- κB luminescent reporter, mechanical testing and histology/biochemistry for collagen and proteoglycan.