Immunotherapy via engineered therapeutic programs in tumors using RNA

免疫疗法治疗，如检查点阻断和嵌合抗原受体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 - 包括诱导免疫原性肿瘤细胞死亡，将免疫细胞募集到肿瘤细胞中， 肿瘤床，以及肿瘤微环境（TME）中免疫抑制因子的逆转。我们有 开发了一种使用肿瘤内施用的合成脂质纳米颗粒（LNP）的治疗方法， 将自我复制（复制子）RNA传递到肿瘤，激活先天免疫信号通路， 表达治疗有效载荷。正如我们最近发表的初步数据所示，这种方法引起了 在几种肿瘤模型中产生了深刻的抗肿瘤免疫应答，并使肿瘤消退， 注射和远端非注射肿瘤。在这里，我们汇集了一个强大的跨学科团队，以这些 初步发现，并应用合成生物学工具包来创建下一代LNP-复制子疗法， 联合收割机的多个功能增加了这种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- 亚基因组启动子复制子，其编码以可调的预定义表达的多个有效负载基因 表达水平，和（3）小分子调控的复制子，允许两阶段治疗方案， 在单次瘤内注射后实施。这些工程化的RNA将与优化的 促进TME中所需靶细胞类型的有效转染的LNP制剂。我们将应用此 技术来治疗癌症死亡的主要原因，肺癌，并评估其影响使用同基因 原位和自体肺癌模型中局部肿瘤内治疗的小鼠模型， 概括了人类肺癌的TME。我们的具体目标是：（1）开发细胞类型的制剂- 在癌细胞和T细胞中特异性表达，（2）创建针对癌症的小分子调节RNA回路 细胞和T细胞用于可编程免疫原性癌细胞死亡和在T细胞中特异性表达。（三） 优化的复制子回路在原位肺癌模型中单独和组合的治疗测试。 总而言之，这项提案汇集了一个高度跨学科的团队，结合了 合成生物学和癌症免疫治疗，以实现更有效，安全和可扩展的形式， 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.