Multi-target suppression of pro-inflammatory cytokines using engineered targeted ribonucleases

使用工程化靶向核糖核酸酶多靶点抑制促炎细胞因子

基本信息

批准号：
10282169
负责人：
SARAH L MICHEL
金额：
$ 42.49万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-15 至 2023-07-14
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10282169
关键词：
3&apos Untranslated Regions Address Animal Model Anti-Inflammatory Agents Binding Biological CCL2 gene CCL3 gene COVID-19 CXCL1 gene Cell model Cells Chimera organism Chimeric Proteins Cleaved cell Cytokine Signaling Cytokine Suppression Development Disease Elements Endoribonucleases Engineering Epithelial Cells Exhibits Family Functional disorder Future Gene Expression Genes Genetic Transcription Goals Guide RNA Human IL8 gene Individual Inflammation Inflammatory Inflammatory Response Influenza Interleukin-1 alpha Interleukin-1 beta Interleukin-18 Interleukin-6 Ions Kinetics Link Lung Measures Mediating Messenger RNA Metals Methods Mitogen-Activated Protein Kinases Modality Modification Organ Pathway interactions Pharmaceutical Preparations Phosphorylation Pilot Projects Population Process Production Property Protein Binding Domain Protein Engineering Proteins RNA RNA Degradation RNA Interference RNA Recognition Motif RNA Sequences Research Respiratory Failure Ribonucleases Sepsis Series Shock Signal Transduction Solubility Specificity Substrate Specificity Syndrome System TIS11 protein TNF gene Technology Testing Therapeutic Viremia Virus Diseases Zinc Fingers airway epithelium base cell type chemokine combinatorial cytokine cytokine release syndrome design endonuclease experimental study high reward high risk improved in vitro activity innovation interest lung injury mRNA Decay mRNA Transcript Degradation macrophage novel therapeutic intervention novel therapeutics patient population programs prototype respiratory virus screening success systemic inflammatory response targeted nucleases targeted treatment tool transcriptome

项目摘要

Cytokine storm syndrome (CSS) is a massive and sustained production of pro-inflammatory cytokines and chemokines triggered by sepsis and severe viral infections including COVID-19 and influenza. This hyper- elevation of cytokine signaling drives the localized and ultimately systemic inflammation responsible for the severe and potentially lethal organ damage associated with this syndrome. There are currently no effective drugs to treat CSS, making development of new therapeutic strategies a top priority. In particular, the limited success observed with approaches targeting individual cytokines indicates that methods are needed that can suppress expression or activity of multiple cytokines simultaneously. To address this need, the goal of this exploratory, high-risk/high-reward R21 proposal is to develop a zinc finger-directed RNA-cleaving agent to suppress pro-inflammatory mRNA subpopulations in cells. Our prototypes link the tandem zinc finger (TZF) domain from tristetraprolin (TTP) to an endoribonuclease domain. This RNA targeting module was selected because it recognizes RNA sequences found in the 3'-untranslated regions of many cytokine and chemokine mRNAs. In cells, chimeric TZF-RNase proteins are expected to bind and rapidly degrade these mRNA substrates, but our design will also allow substrate specificity to be systematically modified. This proposal is aimed at providing the “proof of concept” that TZF-RNase chimeras can function as a deliverable, guided RNA degradation system in cells to suppress a pro-inflammatory gene expression program and production/secretion of associated cytokines. First, we will construct a series of TZF-RNase prototypes and optimize for yield, solubility, and metal ion coordination before functionally screening for sequence-specific RNA cleavage activity in vitro and targeted suppression of candidate pro-inflammatory cytokine mRNAs in cells by accelerating mRNA decay. Second, we will express our optimal TZF-RNase prototype in primary cells relevant to CSS and measure transcriptome-wide effects on mRNA levels and mRNA decay kinetics, followed by effects on cytokine secretion profiles from these cell models. In parallel, we will test methods for delivering TZF-RNase protein into cells. Successful completion of this pilot project will establish proof-of-principle that: (i) an engineered targeted nuclease can post-transcriptionally suppress expression and secretion of multiple pro- inflammatory cytokines associated with CSS, and (ii) that this targeted nuclease can be delivered to and functional in CSS-relevant cell types. Several future applications of this technology are also envisioned, including: (i) discovery tools for characterizing RNA-mediated biological pathways, and (ii) expanding the specificity of the TZF-RNase platform by altering its RNA-targeting specificity. Strategies to broaden the scope include the iterative or combinatorial modification of the TZF moiety and substitution of other RNA-binding domains to `guide' the chimeric protein, creating a tunable family of targeted ribonucleases with long-term impact.

细胞因子风暴综合征（CSS）是促炎细胞因子和由败血症和严重病毒感染引发的趋化因子，包括Covid-19和Intalcene。这个超级细胞因子信号传导的升高驱动局部炎症，最终导致系统性炎症与该综合征有关的严重且潜在的致命器官损伤。目前没有效力治疗CSS的药物，使新的治疗策略的发展成为重中之重。特别是有限用靶向单个细胞因子的方法观察到的成功表明需要方法简单地抑制多种细胞因子的表达或活性。为了满足这一需求，目的是探索性，高风险/高回报R21提案是将锌指的RNA定向剂开发为抑制细胞中的促炎性mRNA亚群。我们的原型连接串联锌指（TZF）从Tristetraprolin（TTP）到核糖核酸酶域的域。选择了此RNA靶向模块因为它识别在许多细胞因子和趋化因子的3'-非翻译区域中发现的RNA序列 mrnas。在细胞中，嵌合TZF-RNase蛋白有望结合并迅速降解这些mRNA 底物，但我们的设计还可以系统地修改底物特异性。该建议旨在提供“概念证明”，即TZF-RNase Chimeras可以作为一个细胞中可交付的，引导的RNA降解系统抑制促炎基因表达程序以及相关细胞因子的生产/分泌。首先，我们将构建一系列TZF-RNASE原型并在功能筛选序列特异性之前优化产量，溶解度和金属离子配位 RNA的裂解活性在体外和靶向抑制细胞中候选促炎细胞因子mRNA 通过加速mRNA衰变。其次，我们将在原代细胞中表达我们的最佳TZF-RNase原型与CSS相关并测量整个转录组对mRNA水平和mRNA衰变动力学的影响，遵循通过对这些细胞模型的细胞因子分泌谱的影响。同时，我们将测试交付的方法 TZF-RNASE蛋白进入细胞。该试点项目的成功完成将确定：（i）工程的靶向核酸酶可以在转录后抑制多个促值的表达和分泌与CSS相关的炎性细胞因子，以及（ii）该靶向核酸酶可以递送到和与CSS相关的细胞类型中的功能。还设想了该技术的几个未来应用，包括：（i）用于表征RNA介导的生物途径的发现工具，以及（ii）扩展 TZF-RNase平台的特异性通过改变其靶向RNA的特异性。扩大范围的策略包括TZF部分的迭代或组合修饰以及其他RNA结合的替代用于“指导”嵌合蛋白的域，形成一个可调的靶向色带菌群家族影响。