Understanding and exploiting muscle and liver contributions to RNA-Nanoparticle immunity

About This Grant

PROJECT SUMMARY mRNA & lipid nanoparticle (RNA-LNP) therapeutics are a versatile new drug modality used for vaccines, gene editing, cell reprogramming, & protein replacement. While vaccines aim to stimulate immunity, other applications must avoid immune activation. Achieving the right immune outcome is critical, as unintended responses can lead to harm or therapeutic failure. Much of the focus of RNA-LNP immunity has been on professional antigen presenting cells (pAPC), such dendritic cells (DC). However, RNA-LNP transfect diverse cell types, including hepatocytes & skeletal myocytes, yet little is known about the role, if any, these cells play in RNA-LNP immunity. To understand better the role of non-pAPCs in the immune response to RNA-encoded protein – with the goal of establishing rules for the design of RNA-LNP that can more precisely & safely control the direction of the immune responses – we generated RNAs that are selectively silenced in different cell populations. Our preliminary data indicate that RNA expression within hepatocytes greatly reduces the quantity of Ag-specific T cells, whereas RNA expression in muscle fibers boosts Ag-specific T cells; establishing that these cells are not bystanders, but active participants, in how RNA-LNP immunity unfolds. We also find that unrestricted RNA expression can lead to hepatic & muscle cell killing when there are pre-existing Ag-specific T cells, and this toxicity can be ameliorated using synthetic target sites for hepatocyte- or muscle-specific miRNA to silence the RNA in these cells. The objective of this project is to elucidate the mechanisms by which RNA-LNP transfection & expression in pAPCs and non-pAPCs, specifically myocytes & hepatocytes, modulate the T cell response to RNA-encoded protein, and to harness these insights to improve the safety and efficacy of cancer and CAR T cell vaccines. In Aim 1, we will determine how RNA expression within hepatocytes dampens T cell response, and test whether silencing RNA cancer vaccines in hepatocytes can enhance tumor immunity & reduce toxicity. In Aim 2, we will test the hypothesis that the T cell response to RNA-LNP is boosted by RNA expression in muscle fibers that serve as a prolonged Ag reservoir that maintains T cell stimulation. In Aim 3, we will test the hypothesis that RNA-LNP transfection of hepatocytes & myocytes turns on stress & infection response pathways that results in RNA-derived antigen being transferred from non-pAPCs to pAPCs for T cell education. These studies will utilize a variety of state-of-the-art models, methods, & technologies, including novel RNA formulations for tailored cell expression, genetic mouse models for immune analysis, spatial transcriptomics for identifying the pathways activated by RNA-LNP in tissues, & normathermic machine perfusion to study RNA-LNP in whole human livers. This project will: (1) help define our understanding of how myocytes & hepatocytes contribute to RNA-LNP immunity, (2) provide novel approaches for modulating the direction of RNA-LNP immunity, and (3) enable more tailored design of RNA cancer vaccines that maximize immune activation & minimize healthy cell killing, which can be used to improve immunotherapies.