Abstract 3472: STX-003: cancer immunotherapy with systemic delivery of mRNA utilizing programmable genetic circuits for precise regulation of IL-12 expression and reduced toxicity

Abstract Introduction: Immunotherapies have revolutionized the treatment of solid tumors, which represent 90% of adult human cancers. Therapeutic delivery of IL-12, a potent immunostimulatory cytokine, is robustly effective in preclinical models. However, systemically delivered IL-12 is poorly tolerated potentially due to its off-target activity. To overcome this challenge, we developed an mRNA platform that utilizes programmable genetic circuits to regulate the expression of an encoded protein in response to microRNAs (miRNAs), enabling precise control of payload expression in the target tissues. Using this platform we developed STX-003, a systemically delivered self-replicating mRNA encoding IL-12 which bears programmable genetic circuitry to limit the expression of payload in off-target tissues while preserving expression in the tumor. Here, we tested the activity of the genetic circuitry as well as its effects on the efficacy and tolerability of systemically delivered IL-12 using preclinical models. Methods: To reduce payload expression in (i.e., detarget) healthy tissues, we first identified unique miRNA signatures using in silico modeling and high-throughput in vitro screening. We then selected candidate circuits, characterized their effectiveness and formulated them in LNPs. Payload expression was tested in vitro using the human cancer cell lines and patient-derived breast cancer organoids. Proof-of-concept studies were conducted in non-human primates and naive mice (with a murine surrogate) to demonstrate successful detargeting of the off-target tissues and its impact on tolerability following systemic delivery. Efficacy was tested in the B16.F10 syngeneic mouse model of melanoma. Tolerability was assessed by monitoring body weights and clinical signs, while efficacy was tracked by measuring tumor volumes over time. Results: STX-003 as well as the uncircuited control expressed the IL-12 payload when transfected into patient-derived tumor organoids and cancer cell lines. Intravenous injection of a murine surrogate of STX-003 improved tolerability and induced anti-tumor responses in a syngeneic mouse model. While STX-003 yielded similar levels of mIL-12 in tumors compared to an uncircuited control, markedly lower levels of mIL-12 were observed in off-target tissues. In healthy non-human primates, systemic administration of STX-003 demonstrated a substantial reduction in circulating IL-12 levels compared to a control without genetic circuitry. Conclusion: These data demonstrate the potential of STX-003, our programmable RNA circuit platform, for expanding the therapeutic index of potent drugs that are limited by systemic toxicities associated with off-target expression. Citation Format: Gautam N. Shenoy, Wen Allen Tseng, Emily A. Clinton, Ryan A. Patenaude, Wuhbet Abraham, Zhijun Su, Madhuri Dey, Justin H. Letendre, Maeve M. Pattie, John T. Sexton, Emily Tjon, Nandita S. Menon, Isma Zarin, Breanna Burgett, Aidan Coia, Akshay Mavani, Weiyu Zhao, Kevin Shi, Alexander C. Lemaire, Julian Quinones, Ryan T. Sowell, Jaspreet S. Khurana, Joseph Barberio, Prashant R. Nambiar, Jacob R. Becraft, Tasuku Kitada. STX-003: cancer immunotherapy with systemic delivery of mRNA utilizing programmable genetic circuits for precise regulation of IL-12 expression and reduced toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3472.