Distinct Inflammatory\nPrograms Underlie the Intramuscular\nLipid Nanoparticle Response

Developments in mRNA/lipid nanoparticle (LNP) technology\nhave advanced\nthe fields of vaccinology and gene therapy, raising questions about\nimmunogenicity. While some mRNA/LNPs generate an adjuvant-like environment\nin muscle tissue, other mRNA/LNPs are distinct in their capacity for\nmultiple rounds of therapeutic delivery. We evaluate the adjuvancy\nof components of mRNA/LNPs by phenotyping cellular infiltrate at injection\nsites, tracking uptake by immune cells, and assessing the inflammatory\nstate. Delivery of 9 common, but chemically distinct, LNPs to muscle\nrevealed two classes of inflammatory gene expression programs: inflammatory\n(Class A) and noninflammatory (Class B). We find that intramuscular\ninjection with Class A, but not Class B, empty LNPs (eLNPs) induce\nrobust neutrophil infiltration into muscle within 2 h and a diverse\nmyeloid population within 24 h. Single-cell RNA sequencing revealed\nSM-102-mediated expression of inflammatory chemokines by myeloid infiltrates\nwithin muscle 1 day after injection. Surprisingly, we found direct\ntransfection of muscle infiltrating myeloid cells and splenocytes\n24 h after intramuscular mRNA/LNP administration. Transfected myeloid\ncells within the muscle exhibit an activated phenotype 24 h after\ninjection. Similarly, directly transfected splenic lymphocytes and\ndendritic cells (DCs) are differentially activated by Class A or Class\nB containing mRNA/LNP. Within the splenic DC compartment, type II\nconventional DCs (cDC2s) are directly transfected and activated by\nClass A mRNA/LNP. Together, we show that mRNA and LNPs work synergistically\nto provide the necessary innate immune stimuli required for effective\nvaccination. Importantly, this work provides a design framework for\nvaccines and therapeutics alike.