PROJECT SUMMARY The innate immune system can be pharmacologically programed to elicit desired immunological outcomes. Retinoic acid-inducible gene I (RIG-I) is a pattern recognition receptor that has emerged as a promising innate immune target for immunopotentiation. RIG-I is activated upon recognizing 5’-triphosphorylated, double- stranded RNA (3pRNA) in the cytosol, which stimulates an antiviral-like inflammatory program that can be harnessed to treat or prevent a diversity of diseases. However, the potency and efficacy of 3pRNA has been limited by major drug delivery barriers, including nuclease degradation, inefficient cellular uptake and cytosolic delivery, and rapid clearance. To address these challenges, we have developed RIG-I activating nanoparticles (RANs). RANs are polymer nanoparticles that are engineered to promote the cytosolic delivery of synthetic, molecularly-defined, and high-affinity stem-loop RNA (SLR) RIG-I agonists recently developed by our team. The objective of this R01 application is to optimize and advance RANs as a versatile platform for pharmacological activation of RIG-I. We will accomplish this through the following Specific Aims. First, we will engineer next-generation RANs with improved properties for systemic administration through optimization of SLR and polymer charge and hydrophobicity. This approach will leverage combinatorial chemical diversity to access a new design space for 3pRNA delivery, which we expect will yield next-generation RANs with higher SLR loading efficiency, reduced cytotoxicity, protection from nuclease degradation, improved stability, and enhanced immunostimulatory activity. Second, we will establish relationships between RAN properties, innate immune activation, pharmacokinetics, polymer and SLR biodistribution, and toxicity. These studies are essential in the preclinical development of new immunotherapeutic modalities and will also yield new insight into how nanocarriers can be engineered for safe and effective activation of RIG-I. We expect these studies to yield next-generation RANs that are optimized for systemic administration of SLR therapeutics. Third, while RANs have broad potential clinical applications, we will evaluate their efficacy as a systemically administered cancer immunotherapy in poorly immunogenic mouse models of melanoma as a clinically important test case. We expect to demonstrate that systemic administration of lead-candidate RANs will activate RIG-I in the tumor microenvironment, resulting in an immunological reprograming of tumor sites that inhibits tumor growth and synergizes with immune checkpoint inhibitors. Collectively, these studies will position RANs as an enabling platform for immunopotentiation with potential to address the significant need for new cancer immunotherapies, antiviral agents, and vaccine adjuvants.

PROJECT NARRATIVE RESEARCH STRATEGY Oligonucleotide agonists of the retinoic acid-inducible gene I (RIG-I) pathway hold significant promise as antiviral agents, vaccine adjuvants, and cancer immunotherapeutics. However, their biological activity and efficacy is limited by poor pharmacological properties, including nuclease degradation, inefficient cellular uptake and cytosolic delivery, and rapid clearance. This proposal aims to address these challenges through the development, optimization, and preclinical evaluation of RIG-I activating nanoparticles (RANs), a platform technology for immunopotentiation via the RIG-I pathway.