1328 STX-003: utilizing programmable genetic circuits for tumor-specific IL-12 expression and reduced toxicity with systemically delivered mRNA

<h3>Background</h3> 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 encoded protein based on specific molecular cues, enabling precise control of payload expression in the target tissues. Using this platform we developed STX-003, a self-replicating mRNA encoding IL-12 which bears programmable genetic circuitry that limits expression of payload in the spleen (the major off-target organ with systemic delivery) while preserving expression in tumors. We tested the activity of this genetic circuit on the efficacy and tolerability of systemically delivered IL-12 using preclinical models. <h3>Methods</h3> To reduce payload expression in (i.e. detarget) the spleen, we first identified unique miRNA signatures using <i>in silico</i> modeling and high-throughput <i>in vitro</i> screening. We then selected candidate circuits, characterized their effectiveness and formulated them in LNPs. The payload expression was tested <i>in vitro</i> using the human lung cancer cell line A549 and patient-derived breast cancer organoids. Proof-of-concept studies were conducted in naive mice and non-human primates to demonstrate successful detargeting of the spleen 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. <h3>Results</h3> STX-003 and the uncircuited control showed similar IL-12 expression levels when transfected into patient-derived tumor organoids and the cancer cell line A549. Intravenous injection of a murine surrogate of STX-003 improved tolerability and induced anti-tumor responses in a syngeneic mouse model. While similar mIL-12 levels were observed in tumors compared to an uncircuited control, markedly lower levels were seen in the spleen. In healthy non-human primates, systemic administration of STX-003 demonstrated a &gt;99% reduction in circulating IL-12 levels compared to a control without genetic circuitry. <h3>Conclusions</h3> These data demonstrate the potential of STX-003, our programmable RNA circuit platform, for the development of precision mRNA therapies for cancer.