ABSTRACT The remarkable effectiveness of the COVID-19 mRNA vaccines heralds a transformative immunization platform against viral infections. A key innovation—recognized with the 2023 Nobel Prize—is the replacement of uridine (U) with N1-methylpseudouridine (m1Ψ) in their mRNA constructs. This substitution reduces side effects and increases antigen production. However, applying m1Ψ-modified mRNA vaccines to the realm of cancer immunotherapy introduces a host of new and complex challenges. These range from understanding the implications of U-to-m1Ψ substitution on anti-tumor CD8+ T cell responses to devising effective priming and boosting strategies, creating more predictive animal models, and surmounting the immunosuppressive elements within the tumor microenvironment (TME). To address these challenges, this proposal outlines a research framework built around mechanistic studies with the goal of generating new mRNA vaccines for pancreatic ductal adenocarcinoma (PDAC)—a cancer with urgent unmet therapeutic needs. Specific Aim 1 seeks to engineer a new class of mRNA vaccines targeting clinically relevant tumor antigens, mesothelin (MSLN), and mutant KRAS (KRASG12D). SubAim 1.1 consists of mechanistic studies to inform strategies for optimizing mRNA-encoded antigen and adjuvant properties and devising effective priming and boosting approaches to enhance immunogenicity and reduce reactogenicity. SubAim 1.2 uses stringent PDAC models to evaluate whether the new vaccines significantly improve the efficacy of T cell transfer therapies. Specific Aim 2 evaluates the new mRNA vaccines in humanized immune system mouse models. Due to significant interspecies differences in innate immune responses to mRNA vaccines, it is vital to move beyond traditional mouse models. SubAim 2.1 aims to understand the effects of these vaccines on human conventional type 1 dendritic cells and subsequent CD8+ T cell activation. SubAim 2.2 focuses on validating the vaccines' safety and efficacy in humanized mouse models engrafted with human PDAC tumors. Specific Aim 3 assesses the potential for allele-specific KRAS inhibitors to reprogram the immunosuppressive PDAC TME, thus enhancing mRNA vaccine efficacy. SubAim 3.1 will investigate whether the new mRNA platform prevents tumor recurrence in PDAC mouse models treated with allele-specific KRAS inhibitors. SubAim 3.2 seeks to elucidate how combining mRNA-based immunotherapies with KRAS-targeted therapies impacts the immunogenicity of PDAC cells, the composition of the immune TME, and anti-tumor efficacy. Deliverables range from developing and optimizing new mRNA vaccines to a systematic mechanistic evaluation of these vaccines in both conventional and humanized mouse models, and finally, to investigating synergies with clinical-stage mutant KRAS-targeted therapies. The anticipated impact consists of advancing the understanding of how new mRNA-based immunotherapies enable priming and sustaining the cancer- immunity cycle and develop...