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

细胞因子风暴综合征(CSS)是一种大量和持续的促炎性细胞因子和 脓毒症和包括新冠肺炎和流感在内的严重病毒感染引发的趋化因子。这个超级- 细胞因子信号的升高会导致局部和最终的全身性炎症 与这种综合征相关的严重的和潜在的致命器官损伤。目前还没有有效的 治疗CS的药物，使开发新的治疗策略成为当务之急。尤其是有限的 通过针对单个细胞因子的方法观察到的成功表明，需要能够 同时抑制多种细胞因子的表达或活性。为了满足这一需求，这一目标 探索性的、高风险/高回报的R21建议是开发一种锌指定向RNA裂解剂来 抑制细胞中的促炎基因亚群。我们的原型连接串联锌指(TZF) 结构域从Tristetraprolin(TTP)到内切核酸酶结构域。该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‘-非翻译区发现的RNA序列 MRNAs。在细胞中，嵌合的TZF-RNase蛋白有望结合并迅速降解这些mRNA 底物，但我们的设计也将允许系统地修改底物特异性。 这项提议旨在提供TZF-RNase嵌合体可以作为 细胞中可传递的、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产生/分泌。首先，我们将构建一系列TZF-RNase原型 并在功能筛选特定序列之前对产率、溶解度和金属离子配位进行优化 RNA体外切割活性及细胞内候选致炎细胞因子mRNAs的靶向抑制 通过加速信使核糖核酸的衰变。其次，我们将在原代细胞中表达我们最优的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，如下 通过这些细胞模型对细胞因子分泌模式的影响。同时，我们将测试交付的方法 TZF-核糖核酸酶蛋白进入细胞。这一试点项目的成功完成将确立以下原则证明：(1) 一种工程化的靶向核酸酶可以转录后抑制多个前-核酸酶的表达和分泌 与CS相关的炎性细胞因子，以及(Ii)该靶向核酸酶可以被递送到和 在与css相关的单元格类型中起作用。还展望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 通过改变TZF-RNase平台的RNA靶向性来提高其特异性。扩大范围的策略 包括TZF部分的迭代或组合修饰和其他RNA结合的替代 结构域来“引导”嵌合蛋白，创建一个可调节的靶向核糖核酸酶家族，具有长期 冲击力。

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.