Development of Programmable mRNA Circuits for Melanoma Immunotherapy

PROJECT SUMMARY While major strides have been made in the development of therapies for melanoma, cases continue to rise, with more diagnoses occurring at an early stage where surgery is indicated for patients. However, even with standard of care anti-PD-1 adjuvant immunotherapy, patients still have a high risk of relapse (3-year relapse-free survival is 50-60%). Neoadjuvant anti-PD-1/anti-CTLA-4 immunotherapy has emerged as a potentially more efficacious alternative to adjuvant immunotherapy, however, widespread usage in these non-metastatic patients would be precluded due to significant toxicities. Therefore, there is a great unmet need to improve outcomes for locally/regionally advanced surgically resectable melanoma patients. Cytokine therapies such as interleukin (IL)- 12 and IL-15 have shown promise in pre-clinical in vitro and in vivo studies, however, when delivered systemically in bolus, their clinical utility is limited due to serious adverse effects as well as suboptimal pharmacokinetics and pharmacodynamics. Thus, we need to find novel ways to locally modulate the immune system early to limit toxicity and reduce the risk of recurrence. To address this challenge in melanoma in this NIH Phase I SBIR, Strand Therapeutics is proposing to engineer a tunable and programmable small molecule-regulated self-replicating mRNA (repRNA)-based combinatorial cytokine immunotherapy that mimics the physiological expression kinetics of IL-12 (expressed early) and IL-15 (expressed later). In doing so, this programmable repRNA immunotherapy is designed to enhance the patient’s own immune system to combat melanoma tumors and provide durable immune surveillance and remission with limited toxicity. In Aim 1, we will construct the mRNA circuit and validate expression kinetics of our programmed mRNAs using surrogate luciferase reporters, which will allow us to assess in vivo expression kinetics in real-time in a mouse model of melanoma. In Aim 2, we will encode IL-12 and IL-15 in the circuit validated in Aim 1 and assess expression kinetics and tunability of IL-12/IL-15 expression in a mouse model of melanoma. In Aim 3, we will test if our engineered circuit can eliminate tumors in vivo in mouse models of melanoma, and benchmark against non- circuit IL-12/IL-15 delivery approaches such as recombinant cytokines and constitutive expression from mRNAs. Successful completion of these studies will lead to a novel programmable circuit with IL-12/IL-15 for the treatment of melanoma. Through this project, we intend to program the natural kinetics of IL-12/IL-15, which will induce stronger and longer-lasting anti-cancer immune responses and increase the efficacy of anti-PD-1/PD-L1 therapies, without the side-effects linked to systemically delivered cytokines.PROJECT NARRATIVE While there have been major strides in the development of therapies for melanoma, the number of cases is increasing, with more diagnoses occurring at an early stage where surgery is indicated; however, locally advanced patients are still at a high risk of relapse following surgery. To address this unmet need, Strand Therapeutics is engineering a programmable mRNA circuit with temporal expression of cytokines, interleukin-12 (IL-12) and interleukin-15 (IL-15), for effective stimulation of the adaptive immune response as a neoadjuvant therapy for melanoma. Our proprietary lipid nanoparticles will be used to deliver self-replicating mRNA, which will temporally express IL-12 and IL-15 directly to the tumor microenvironment (TME), to increase TME immunogenicity and increase the efficacy of anti-PD-1 therapy, without the side effects linked to systemically delivered cytokines.