PROJECT SUMMARY Triple negative breast cancer (TNBC) has few treatments, and patients have a poor prognosis. Checkpoint inhibitors (e.g., anti-PD-1 antibodies) represent a promising new therapeutic strategy, and two recent clinical trials demonstrate that TNBC patients may benefit. However, only a fraction of patients responded indicating that additional combination therapy with anti-PD-1 is needed. Strand Therapeutics developed a synthetic self- replicating mRNA (repRNA) for use with anti-PD-1. We achieve tumor-directed expression by encapsulating and directly injecting the in vitro transcribed repRNA with our fully developed lipid nanoparticle (LNP) into the tumor. The entire repRNA manufacturing process is done in vitro, giving us advantages over virus-based approaches that require mammalian cell culture. Once in the tumor microenvironment (TME), the repRNAs express a potent engineered form of the interleukin 12 (IL-12) fusion protein (IL-12-lumican) to stimulate the anti-tumor activity of NK cells and CD8 T cells in TNBC lesions. The IL-12-lumican fusion protein reduces the risk of systemic toxicity and enhanced activity by retaining IL-12 within the TME via a collagen-binding domain (lumican). In addition, repRNA expression and replication have advantages over the short expression duration of modified RNA (modRNA), and repRNA stimulates various pattern recognition receptors such as TLRs, RIG-I, and MDA-5 to upregulate type-I interferon. Furthermore, Strand Therapeutics’ proprietary LNP not only efficiently delivers mRNA to cells but also uniquely synergizes with the repRNA to induce oncolytic effects (immunogenic cell death). These additional inflammatory cues facilitate turning a “cold” TME to one that will respond to anti-PD-1 therapy. In this proposal, we will determine whether tumoral injection of repRNA IL-12-lumican can effectively overcome insufficient immune activation in the TME and augment responses to anti-PD-1 therapy in TNBC. In Aim 1 we will demonstrate that in TNBC cells repRNAs have more sustained transgene expression and greater type I IFN induction than modRNAs. In Aim 2 we will demonstrate that repRNAs in human cell line-derived xenograft (CDX) and patient-derived xenografts (PDX) have greater sustained expression than modRNAs. In Aim 3 we will demonstrate efficacy and superiority of repRNA IL-12-lumican over modRNA IL-12-lumican treatment (in combination with anti-PD-1) in vivo in syngeneic mouse models of TNBC and ex vivo in primary explants from TNBC patients. These studies will provide proof of concept data that Strand Therapeutics’ approach using self- replicating mRNA encoding IL-12 in combination with PD-1 antibody blockade is effective at reducing TNBC tumor volume.