Immunotherapy via engineered therapeutic programs in tumors using RNA

免疫疗法，如检查点封锁和嵌合抗原受体T细胞疗法 证明了免疫系统根除转移性癌症的能力，但 实体肿瘤的免疫治疗仍然局限于少数患者。一系列相互关联的事件是 疗效所需-包括诱导免疫原性肿瘤细胞死亡，将免疫细胞招募到 肿瘤床，以及肿瘤微环境(TME)中免疫抑制信号的逆转。我们有 开发了一种使用肿瘤内注射合成脂质纳米粒(LNPs)的治疗方法 将自我复制(复制子)RNA传递给肿瘤，激活先天免疫信号通路并有效地 快速治疗有效载荷。正如我们最近公布的初步数据所显示的那样，这种方法引发了 在几种肿瘤模型中产生深刻的抗肿瘤免疫反应，并使两种肿瘤模型的肿瘤消退 注射肿瘤和远端未注射肿瘤。在这里，我们汇聚了一支强大的跨学科团队，以此为基础 初步发现并应用合成生物学工具包来创造下一代LNP复制子疗法，该疗法 结合多种功能以提高该方法的安全性和有效性，包括：(1)细胞分类器 仅允许复制子在靶癌细胞或免疫细胞中表达的电路，(2)优化的多 编码多种有效载荷基因的亚基因组启动子复制子 表达水平，以及(3)小分子调控的复制子，允许两阶段治疗计划 在单次肿瘤内注射后实施。这些经过改造的RNA将与优化的 在TME中促进所需靶细胞类型的高效转染的LNP制剂。我们将应用这一点 治疗癌症死亡的主要原因肺癌并使用同源基因评估其影响的技术 小鼠原位和原位肺癌局部瘤内治疗模型的建立 重述人类肺癌的TME。我们的具体目标是：(1)开发针对细胞类型的配方- 在癌细胞和T细胞中的特异性表达，(2)为癌症创造小分子调节的RNA电路 细胞和T细胞对免疫原性癌细胞的程序性死亡和在T细胞中的特异性表达。(3) 在原位肺癌模型中单独和联合应用优化复制子电路的治疗测试。 总而言之，这项提议汇集了一个高度跨学科的团队，将来自 合成生物学和癌症免疫疗法，以实现更有效、安全和可扩展的 免疫疗法。

Immunotherapy treatments such as checkpoint blockade and chimeric antigen receptor T cell therapy have demonstrated the power of the immune system to eradicate metastatic cancer, but the efficacy of immunotherapies in solid tumors remains confined to a minority of patients. A series of interlinked events are needed for efficacy – including induction of immunogenic tumor cell death, recruitment of immune cells to the tumor bed, and reversion of immunosuppressive cues in the tumor microenvironment (TME). We have developed a therapeutic approach using intratumorally-administered synthetic lipid nanoparticles (LNPs) to deliver self-replicating (replicon) RNAs to tumors that activate innate immune signaling pathways and potently express therapeutic payloads. As shown in our recently published preliminary data, this approach elicited profound anti-tumor immune responses in several tumor models, and enabled tumor regression of both injected and distal non-injected tumors. Here we bring together a strong interdisciplinary team to build on these initial findings and apply a synthetic biology toolkit to create next-generation LNP-replicon therapeutics, which combine multiple features to increase the safety and efficacy of this approach, including: (1) cell classifier circuits that allow replicon expression only in target cancer cells or immune cells, (2) optimized multi- subgenomic promoter replicons that encode multiple payload genes expressed at tunable predefined expression levels, and (3) small molecule-regulated replicons that allow two-stage therapeutic programs to be implemented following a single intratumoral injection. These engineered RNAs will be combined with optimized LNP formulations that promote efficient transfection of desired target cell types in the TME. We will apply this technology to treat the leading cause of cancer death, lung cancer, and assess its impact using a syngeneic mouse model of local intratumoral therapy in orthotopic and autochthonous lung cancer models that recapitulate the TME of human lung cancers. Our specific aims are: (1) Develop formulations for cell type- specific expression in cancer cells and T cells, (2) Create small molecule-regulated RNA circuits for cancer cells and T cells for programmable immunogenic cancer cell death and specifically expression in T cells. (3) Therapeutic testing of optimized replicon circuits in orthotopic lung cancer models alone and in combination. Altogether, this proposal brings together a highly interdisciplinary team, marrying cutting edge concepts from synthetic biology and cancer immunotherapy to achieve a more effective, safe, and scalable form of immunotherapy.