Synthetic Cytosolic Splicing Enables Programmable mRNA-Encoded Receptors

Abstract

A typical cell therapy product comprises engineered cells that detect disease-related molecular cues in their surrounding or on a surface of a target cell, and activate a response that alleviates disease symptoms or eradicates diseased cells. mRNA as a therapeutic substrate has become prevalent in the last decade across multiple therapeutic areas, and it has also been evaluated as a building block of cell therapies. However, compared to DNA-based building blocks, it is much more challenging to use mRNA in a programmable manner to engineer complex multi-input/multi-output processes that can fully support the next generation of cell and gene therapies. Addressing this challenge requires the exploration of novel post-transcriptional control mechanisms that bridge mRNA regulation with extracellular surroundings. Here, we engineer a family of synthetic mRNA splicing (SMS) receptors by redesigning the Inositol-requiring enzyme 1 (IRE1) to regulate protein synthesis from a precursor mRNA. We design SMS-based receptors that sense diverse intracellular and extracellular inputs, highlighting the versatility and modularity of this platform. We apply this approach to design a cytokine-converter receptor that detects inflammatory cytokines and produces an anti-inflammatory output in response. That receptor is successfully validated in cell lines and primary T cells upon mRNA delivery. These cells generate anti-inflammatory IL-10 upon stimulation by physiological levels of either TNF- or IL-1{beta} secreted by macrophage-like cells, highlighting their potential as a cell therapy for inflammatory diseases. With its modular and programmable architecture, the SMS platform is poised to become an important enabling tool for sophisticated programmable mRNA therapeutics.