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

细胞因子风暴综合征（CSS）是一种大量和持续产生的促炎细胞因子， 败血症和严重病毒感染（包括COVID-19和流感）引发的趋化因子。这个超- 细胞因子信号传导的升高驱动了局部的和最终的全身性炎症， 严重和潜在致命的器官损伤与这种综合征。目前还没有有效 治疗CSS的药物，使新的治疗策略的发展成为当务之急。特别是有限的 用靶向单个细胞因子的方法所观察到的成功表明需要能够 同时抑制多种细胞因子表达或活性。为了满足这一需求， 探索性的，高风险/高回报的R21建议是开发一种锌指导向的RNA切割剂， 抑制细胞中的促炎mRNA亚群。我们的原型将串联锌指（TZF） 在一个实施方案中，所述多肽包含从三曲脯氨酸（TTP）到核糖核酸内切酶结构域的结构域。这个RNA靶向模块被选中 因为它识别在许多细胞因子和趋化因子的3 '-非翻译区中发现的RNA序列 mRNA。在细胞中，预期嵌合TZF-RNase蛋白结合并快速降解这些mRNA 我们的设计也将允许系统地修改底物特异性。 该提案旨在提供TZF-RNase嵌合体可以作为一种免疫调节剂发挥作用的“概念证明”。 细胞中可递送的引导RNA降解系统，以抑制促炎基因表达程序 和相关细胞因子的产生/分泌。首先，我们将构建一系列TZF-RNase原型 并在功能性筛选序列特异性之前优化产率、溶解度和金属离子配位。 体外RNA切割活性和细胞中候选促炎细胞因子mRNA的靶向抑制 通过加速mRNA的衰变。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，并测量对mRNA水平和mRNA衰减动力学的全转录组效应， 通过对这些细胞模型的细胞因子分泌谱的影响。与此同时，我们将测试交付方法， TZF-RNase蛋白进入细胞。该试点项目的成功完成将确立以下原则证明： 工程化的靶向核酸酶可以在转录后抑制多个前体的表达和分泌， 与CSS相关的炎性细胞因子，和（ii）这种靶向的核酸酶可以被递送到并 在CSS相关细胞类型中起作用。还设想了该技术的若干未来应用， 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 通过改变TZF-RNase平台的RNA靶向特异性来增强TZF-RNase平台的特异性。扩大范围的战略 包括TZF部分的反复或组合修饰和其它RNA结合部分的取代 结构域来“引导”嵌合蛋白，产生一个可调的靶向核糖核酸酶家族， 冲击

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.