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 domai...