Development of Programmable mRNA Circuits for Melanoma Immunotherapy

项目摘要 虽然黑色素瘤治疗的发展取得了重大进展，但病例继续上升， 更多的诊断发生在需要手术的早期阶段。然而，即使标准 尽管接受抗PD-1辅助免疫治疗，患者仍有较高的复发风险（3年无复发生存期 50-60%）。新辅助抗PD-1/抗CTLA-4免疫疗法已成为一种潜在的更有效的免疫疗法。 然而，作为辅助免疫疗法的替代，在这些非转移性患者中的广泛使用将是 由于严重的毒性而被排除。因此，改善结果的需求很大， 局部/区域晚期可手术切除的黑色素瘤患者。细胞因子治疗，如白细胞介素（IL）- IL-12和IL-15在临床前体外和体内研究中显示出前景，然而，当全身递送时， 在推注中，由于严重副作用以及次优药代动力学， 药效学因此，我们需要找到新的方法来早期局部调节免疫系统， 毒性，降低复发风险。为了解决NIH I期SBIR中黑色素瘤的这一挑战， Strand Therapeutics公司正在设计一种可调和可编程的小分子调节的 基于自我复制mRNA（repRNA）的组合细胞因子免疫疗法， IL-12（早期表达）和IL-15（晚期表达）的生理表达动力学。在这样做时， 可编程repRNA免疫疗法旨在增强患者自身的免疫系统， 黑色素瘤肿瘤，并提供持久的免疫监视和缓解有限的毒性。在目标1中，我们 构建mRNA回路，并使用替代物验证我们编程的mRNA的表达动力学 荧光素酶报告基因，这将使我们能够在小鼠模型中实时评估体内表达动力学， 黑素瘤在目标2中，我们将在目标1中验证的电路中编码IL-12和IL-15，并评估表达 黑色素瘤小鼠模型中IL-12/IL-15表达的动力学和可调性。在目标3中，我们将测试我们的 工程电路可以消除体内黑色素瘤小鼠模型中的肿瘤，并以非肿瘤细胞为基准。 循环IL-12/IL-15递送方法，例如重组细胞因子和来自mRNA的组成型表达。 这些研究的成功完成将导致一种新的可编程电路与IL-12/IL-15的治疗 黑色素瘤通过这个项目，我们打算对IL-12/IL-15的自然动力学进行编程，这将诱导 更强和更持久的抗癌免疫应答，并增加抗PD-1/PD-L1的功效 治疗，没有与全身递送的细胞因子相关的副作用。

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.