PROJECT SUMMARY In the first 6 months after COVID-19 vaccine approval, 3.4 billion doses were administered globally, potentially saving 7.2 million lives. Lipid nanoparticle (LNP)-mRNA based COVID-19 vaccines have been able to induce both a strong T cell- and antibody-mediated responses against SARS-CoV-2 by triggering myeloid antigen presenting cells (APCs) such as dendritic cells (DCs), demonstrating the efficacy of LNP-mRNA technology to modulate the immune system. Partly due to the success of COVID-19 vaccines, there has been a renewed attention in engineering novel LNP formulations to better modulate the myeloid APC compartment by 1) either fine-tuning the adjuvanticity of the LNP lipid formulation or 2) improving in vivo targeting efficiency of LNPs towards myeloid APCs. Although the advancements in LNP lipid chemistry enrich our arsenal of LNP formulations suited to modulate the immune system, there hasn't been a corresponding detailed understanding of the molecular and cellular changes induced upon the uptake of LNPs in specific cell types and tissues. In fact, attempts to elucidate the innate mechanisms of LNP immunogenicity have often led to conflicting conclusions and have in turn suggested that the mechanisms of LNP immunogenicity are rather LNP formulation-dependent. The lack of consensus on the innate mechanisms of LNP immunogenicity further highlight that the immunogenicity of each LNP is a complex function of a) its lipid formulation and the nature of its payload, b) the pattern recognition receptor (PRR) profile unique to each immune subset, c) LNP targeting efficiency in each cell type and d) cell, tissue and disease context of LNP treatment. Without the parallel efforts to gain mechanistic understanding of immunogenicity of each LNP formulation, it remains difficult to rationally select LNP formulations that are tailored to a particular disease context, including cancer. In using LNPs as a cancer immunotherapeutic, my central hypothesis states that LNP lipid formulations that drive up their immunogenicity combined with an engineering strategy to increase specific targeting of DCs in vivo can synergize with immunostimulatory payloads (i.e. IL-12) to reprogram DC cell states and enhance anti-tumor immunity. In Aim 1, I will characterize the changes in DC phenotype, function and transcriptional profile upon uptake of DC-targeted LNPs in both steady state and tumoral contexts using DC-T cell co-cultures, bulk and scRNA-seq of sorted LNP transfected DCs. In Aim 2, I will engineer Clec9a targeted bi-specific antibodies to improve LNP transfection of DCs in vivo. The outcome of this project is both a mechanistic understanding of the interplay between LNPs and DCs that occurs upon LNP uptake and a novel bi-specific antibody-based strategy to increase DC targeting in vivo.