PROJECT SUMMARY: The generation of a robust disease-specific immune response is a critical challenge in cancer immunotherapies, many of which provide clinical benefit in only a fraction of patients despite remarkable successes, and in infectious disease vaccines. Harnessing the innate immune system, which has evolved to sense and respond to complex combinations of pathogen-associated molecular patterns, is highly promising for augmenting cancer immunotherapies and infectious disease vaccines. While many synthetic innate immune agonists that activate pattern recognition receptors (PRRs) have been developed as vaccine adjuvants, only a handful of adjuvants have been clinically approved. Generation of a durable immune response faces key barriers: (1) many current PRR agonist-based adjuvants rely on a single agonist, poorly recapitulating natural pathogen recognition by the innate immune system; (2) while mounting evidence shows that combining PRR agonists can promote synergistic activation, control and understanding of the combinatorial effects and spatial modulation of multiple agonists remain inadequate; and (3) further exacerbating these gaps is a scarcity of platforms for precisely tuning combinations and spatial arrangements of innate immune agonists. To address these knowledge and technological gaps, the proposed work will engineer a molecularly defined, modular “all-in-one” platform that utilizes RNA as both a multifunctional agonist and scaffold to sculpt the immune response via (1) precise spatial patterning and multivalency of PRR agonists; (2) combinatorial control of innate immune signaling pathways; and (3) targeted self-delivery. The first aim will leverage the programmable structural features and sequence of a modular RNA scaffold to combine and pattern PRR agonists for synergistic innate immune activation. In an orthogonal aim, the team will exploit the natural immune cell-targeting abilities of fungal wall polysaccharides to create a bioinspired, “all-in-one” synthetic glyco-RNA that can target and activate antigen-presenting cells. The translational potential of the developed RNA platform will then be explored in a proof-of-concept evaluation of in vivo therapeutic efficacy in the third aim, using a syngeneic mouse model of melanoma. If successful, the proposed work will introduce a new paradigm for the rational design of combinatorial innate immune agonists and a new class of synthetic glyco-RNA therapeutic molecules for targeted immune activation. The highly modular platform will enable (1) fundamental studies of how combinations of innate immune agonists and their spatial arrangements function at the molecular, cellular, and organismal levels; and (2) precise modulation of disease-specific immune responses for applications in cancer and infectious diseases.