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

> **NIH NIH R21** · UNIVERSITY OF MARYLAND BALTIMORE · 2021 · $424,875

## Abstract

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...

## Key facts

- **NIH application ID:** 10282169
- **Project number:** 1R21EB032019-01
- **Recipient organization:** UNIVERSITY OF MARYLAND BALTIMORE
- **Principal Investigator:** SARAH L MICHEL
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $424,875
- **Award type:** 1
- **Project period:** 2021-07-15 → 2023-07-14

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10282169

## Citation

> US National Institutes of Health, RePORTER application 10282169, Multi-target suppression of pro-inflammatory cytokines using engineered targeted ribonucleases (1R21EB032019-01). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10282169. Licensed CC0.

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