Immunotherapy treatments such as checkpoint blockade and chimeric antigen receptor T cell therapy have demonstrated the power of the immune system to eradicate metastatic cancer, but the efficacy of immunotherapies in solid tumors remains confined to a minority of patients. A series of interlinked events are needed for efficacy – including induction of immunogenic tumor cell death, recruitment of immune cells to the tumor bed, and reversion of immunosuppressive cues in the tumor microenvironment (TME). We have developed a therapeutic approach using intratumorally-administered synthetic lipid nanoparticles (LNPs) to deliver self-replicating (replicon) RNAs to tumors that activate innate immune signaling pathways and potently express therapeutic payloads. As shown in our recently published preliminary data, this approach elicited profound anti-tumor immune responses in several tumor models, and enabled tumor regression of both injected and distal non-injected tumors. Here we bring together a strong interdisciplinary team to build on these initial findings and apply a synthetic biology toolkit to create next-generation LNP-replicon therapeutics, which combine multiple features to increase the safety and efficacy of this approach, including: (1) cell classifier circuits that allow replicon expression only in target cancer cells or immune cells, (2) optimized multi- subgenomic promoter replicons that encode multiple payload genes expressed at tunable predefined expression levels, and (3) small molecule-regulated replicons that allow two-stage therapeutic programs to be implemented following a single intratumoral injection. These engineered RNAs will be combined with optimized LNP formulations that promote efficient transfection of desired target cell types in the TME. We will apply this technology to treat the leading cause of cancer death, lung cancer, and assess its impact using a syngeneic mouse model of local intratumoral therapy in orthotopic and autochthonous lung cancer models that recapitulate the TME of human lung cancers. Our specific aims are: (1) Develop formulations for cell type- specific expression in cancer cells and T cells, (2) Create small molecule-regulated RNA circuits for cancer cells and T cells for programmable immunogenic cancer cell death and specifically expression in T cells. (3) Therapeutic testing of optimized replicon circuits in orthotopic lung cancer models alone and in combination. Altogether, this proposal brings together a highly interdisciplinary team, marrying cutting edge concepts from synthetic biology and cancer immunotherapy to achieve a more effective, safe, and scalable form of immunotherapy.

In this application, we propose to use synthetic biology approaches to engineer self- replicating RNAs as powerful anti-cancer therapeutics. Our RNA-based synthetic gene circuits will provide a new paradigm for cancer immunotherapy of diverse cancers.