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

序列特异性 CRISPR 介导的炎症细胞因子受体调节用于治疗炎症性椎间盘病理学

• 批准号: 9284378
• 负责人: Robert D. Bowles
• 金额: $ 7.45万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-03 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9284378
• 关键词: Alginates; Anti-Inflammatory Agents; Anti-inflammatory; Back Pain; Base Sequence; Biochemistry; CRISPR interference; Cell Count; Cell Line; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Collagen; Complementary RNA; Cytokine Network Pathway; Cytokine Receptors; Development; Disease; Down-Regulation; Enzymes; Event; Extracellular Matrix Degradation; Flow Cytometry; Gene Activation; Gene Expression; Gene Expression Regulation; Genes; Genome engineering; Genomics; Goals; Guide RNA; Healthcare; Histology; Human; Hydrogels; IL1R2 gene; Immune; Inflammation; Inflammatory; Inflammatory Response; Interleukin-1 beta; Interleukin-6; Intervertebral disc structure; Lentivirus Vector; Link; Low Back Pain; Mammalian Cell; Measures; Mechanics; Mediating; Methods; Neurons; Nociception; Operative Surgical Procedures; Pain; Pathogenesis; Pathologic; Pathology; Patients; Population; Process; Proteoglycan; Radiculopathy; Regulation; Reporter; Research; Role; Secondary to; Signal Transduction; Site; Specificity; Spinal Fusion; System; TNF gene; TNFRSF1A gene; TNFRSF1B gene; Techniques; Technology; Testing; Therapeutic; Tissue Banks; Tissues; Up-Regulation; Vertebral column; base; burden of illness; cell type; cellular transduction; cytokine; design; disability; disability-adjusted life years; improved; inflammatory milieu; intervertebral disk degeneration; knock-down; macrophage; novel; novel therapeutics; nuclease; promoter; public health relevance; receptor; response; tool; vector

 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.

 描述（由申请人提供）：椎间盘（IVD）病理学和相关背痛是一个主要的医疗保健问题，在疾病负担（残疾调整生命年）中排名第三。炎症和相关的促炎细胞因子（例如TNF-α、IL-1 β、IL-6、IL-8）已经作为活性组分参与与背痛相关的许多事件和过程，包括椎间盘退变、IVD疝出以及神经根病和退变椎间盘中的伤害感受神经元的致敏。因此，这种细胞因子网络是腰痛治疗的主要靶标。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剂。使用CRISPR系统抑制和激活多个基因的策略将提供一种强大的工具来拮抗在IVD病理学中活跃的冗余促炎细胞因子网络。CRISPR调控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。该计划旨在开发基于CRISPR的炎症信号调节在退行性椎间盘中的新用途，并测试其作为治疗策略的潜力。在目标1中，我们将测试CRISPR基因调控系统可以在功能上调节退行性椎间盘中发现的细胞类型中的炎症反应的假设。在目标1A中，我们将开发基于CRISPR的序列特异性下调（CRISPRi），用于椎间盘退变中关键炎症细胞因子受体的基因表达。将开发表达dCAS-KRAB和靶向TNF-α、IL-1β和IL-6受体的向导RNA的慢病毒载体（即TNFR 1 i、IL 1 R1 i、Il 6 Ri、Il 6STi），并筛选其在调节人NP细胞、人DRG神经元和人巨噬细胞中受体呈递方面的功效。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）。将开发表达dCAS-VP 64和靶向Il 1 R2和TNFR 2的向导RNA的慢病毒载体，并评价其在NP细胞、人DRG神经元和人巨噬细胞中的疗效。在目标1A和1B中，将使用qPCR和NF-κB报告基因测量基于CRISPR的受体调节后细胞对炎性细胞因子暴露的应答。在目标2中，我们将测试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功能性IVD组织发育的假设。我们将使用基于CRISPR的序列特异性多重调节炎症受体来拮抗IVD细胞和组织中炎症的有害退行性作用。将通过qPCR、NF- κB发光报告基因、胶原蛋白和蛋白聚糖的机械试验和组织学/生物化学评估炎症条件下的功能性IVD组织发育和细胞反应。

项目成果

CRISPR Epigenome Editing of AKAP150 in DRG Neurons Abolishes Degenerative IVD-Induced Neuronal Activation.

• DOI: 10.1016/j.ymthe.2017.06.010
• 发表时间: 2017-09
• 期刊: Molecular therapy : the journal of the American Society of Gene Therapy
• 作者: J. Stover;Niloofar Farhang;Kristofer C. Berrett;Jason Gertz;B. Lawrence;R. Bowles