Tissue-specific silencing of synthetic mRNAs by de-targeting elements maps vaccination-competent tissues and allows Cas9 de-immunization

The clinical success of mRNA/lipid nanoparticle (LNP)-based vaccines has established a leading platform for in vivo nucleic acid delivery, yet the relationship between biodistribution, tissue-specific translation, and immune activation remains poorly defined. Here, we systematically investigate the anatomical determinants of immune responses elicited by mRNA/LNPs. Using a gold-standard LNP formulation, we map exogenous mRNA biodistribution and functionally interrogate the contribution of distinct organs to both humoral and cellular immunity. To achieve this, we engineered mRNAs containing 3' UTR-embedded de-targeting elements as target sites for endogenous tissue-specific microRNAs, enabling selective silencing of antigen expression in defined anatomical compartments. This approach allowed us to causally link sites of translation with immune outputs. Applying this strategy to a spike-based vaccine model, we identify muscle and spleen as the most immunologically competent tissues required for robust immune priming. These findings provide a framework for the rational design of next-generation mRNA/LNP formulations with enhanced targeting precision, enabling dose sparing and reduced off-target effects. Furthermore, we extended this concept to genome editing by preventing mRNA-Cas9 expression in immune-active tissues, thereby reducing its immunogenicity and toxicity. Collectively, our work establishes a unifying strategy to optimize both potency and safety of mRNA-based therapeutics.