Development of an RNA system to tailor transgene expression pattern in vivo and study how different cell types contribute to RNA vaccine immune response.

Modified RNA-based drugs (modRNA) are emerging as a powerful new class of medicine. Already one type of modRNA, RNA vaccines, have helped immunize against SARS-CoV-2. modRNA is delivered via lipid carriers and whichever cell takes up and expresses the modRNA can present antigen. The identity & state of cells presenting antigen determine whether cognate T cells acquire effector or tolerogenic phenotype. While dendritic cells (DCs) were shown to be important for priming of RNA vaccine immunity, the influence and contributions of other transfected cell types to the immune response to modRNA-encoded proteins are not known. Because this contribution could actually oppose the therapeutic goal, for instance promoting tolerance instead of clearance of the pathogen, it is fundamental to answer this question. Professor Brown's laboratory previously developed a technology that enables highly effective silencing of vectorexpressed RNA in desired cells. They showed that incorporation of synthetic microRNA target sites (miRT) into a transgene RNA can prevent its translation in cells expressing the cognate microRNA (miRNA). The objective of this project is to establish miRT as a broad platform to tailor modRNA cell expression patterns and to apply miRT to determine how modRNA expression in specific cells impacts immunity to modRNA-encoded proteins. In this project we will use the latest miRNA atlas data & understanding of miRNA control rules to rationally design different modRNA.miRT formulations for tailored expression of modRNA from different cell types in vivo, including myocytes, endothelial cells, & fibroblasts. If successful, these targeted formulations will have broad translational applications to the future of modRNA vaccines and therapeutics. We will use miRT to silence modRNA in specific cell types in mice. Then we will assess abundance and phenotype of antigen-specific T & B cell and antibody (Ab) responses to model antigen & spike protein using ELISA, FACS and multiplex imaging. These studies will provide a means to alter the immune response to antigens encoded by modRNA. They will also provide mechanistic insight into the role of different cells in the outcome of modRNA vaccination.