Reprogramming the tumor microenvironment via self-amplified RNA (SafeR) circuits

 描述：来自不同癌症类型患者的数据显示，肿瘤中免疫细胞浸润的增加与患者预后的改善相关。同样清楚的是，肿瘤产生大量免疫抑制信号以阻止免疫攻击，因此内源性免疫反应在大多数情况下无法消除已建立的肿瘤。最近，通过“检查点阻断”药物阻断T细胞上的单个抑制信号受体来改变肿瘤中抑制信号的努力已经显示出良好的临床结果，表明肿瘤通过使肿瘤微环境向免疫前状态转变而消退。然而，阻断单个信号通路很可能会 癌症免疫治疗不足以充分发挥其潜力，因为肿瘤产生了一个复杂的抑制信号网络。在这里，我们提出了一种基于合成生物学和系统生物学相结合的方法，通过向肿瘤微环境中的细胞传递复杂的多步遗传电路来重新编程肿瘤中的抑制性微环境。这一策略将通过基于自我复制的RNA电路的开发来实现，该电路可以(I)通过miRNA表达谱识别肿瘤中特定的细胞类型，(Ii)利用小分子调节的诱导新的基因表达程序来作用于肿瘤中的周围细胞，以及(Iii)以协调的方式改变已识别细胞的功能，以改变肿瘤内的平衡，从免疫抑制转向免疫介导的肿瘤破坏。我们的具体目标是：(1)设计一个包含RNA结合调控蛋白和小分子诱导蛋白降解结构域的体内RNA合成生物学平台；(2)我们将分析4T1乳腺癌细胞和B16F10黑色素瘤细胞的miRNA特征，创建仅允许复制子在靶细胞类型中表达的RNA编码的多输入分类电路；(3)我们将建立一系列日益复杂的程序化癌症治疗方法，其中细胞因子的分泌和坏死基因的表达仅限于肿瘤细胞并且可以由FDA批准的小分子进行精确调控，以及安全开关机制，以消除健康细胞中的复制子编码电路。我们将把我们的治疗电路传递到体内的小鼠肿瘤中，并监测抗癌免疫反应，使用系统生物学原理来分析肿瘤中多细胞网络的结果反应。这些研究的一个关键目标是设计RNA电路，以驱动转基因的肿瘤/免疫细胞在微环境中对周围细胞起反式作用，实现肿瘤范围内的肿瘤环境变化，而不需要成功地将电路传递到肿瘤中的每一个细胞。该项目的结果将为设计哺乳动物细胞功能提供一个基于Ne RNA的工具包，展示这些方法在体内的实用性，并为克服传统化疗和靶向药物治疗的局限性的肿瘤重新编程提供一个框架。

 DESCRIPTION: Data from patients in diverse cancer types show that increased immune cell infiltration of tumors correlates with improved patient prognosis. It is also clear that a host of immunosuppressive signals are produced by tumors in order to block immune attack, and thus the endogenous immune response is in most cases unable to eliminate established tumors. Recently, efforts to alter the suppressive signaling in tumors through "checkpoint blockade" drugs that block individual suppressive signaling receptors on T-cells have shown promising clinical results, demonstrating tumor regression through shifting of the tumor microenvironment toward a pro-immunity state. However, it is likely that blocking individual signaling pathways will be insufficient for cancer immunotherapy to reach its full potential, because tumors create a complex network of suppressive signals. Here we propose an approach based on integration of synthetic biology and systems biology to reprogram the suppressive microenvironment in tumors through delivery of sophisticated multi-step genetic circuits to cells in the tumor microenvironment. This strategy will be enabled by the development of self-replicating RNA-based circuits that can (i) identify cell types specifically in the tumor through miRNA expression profiles, (ii) utilize small molecule-regulated induction of new gene expression programs that act in trans on surrounding cells in the tumor, and (iii) alter the function of identified cells in a coordinated fashion to tip the balance within the tumor from immune suppression to immune-mediated tumor destruction. Our specific aims are: (1) We will design a platform for in vivo RNA-based synthetic biology comprising RNA-binding regulatory proteins and small molecule induced protein degradation domains; (2) We will profile the miRNA signatures of 4T1 breast cancer cells and B16F10 melanoma cancer cells and create RNA-encoded multi-input classifier circuits that permit replicon expression only in target cell types, and (3) We will build a series f increasingly sophisticated programmed cancer therapies where cytokine secretion and necroptotic gene expression is restricted to tumor cells and can be precisely regulated by FDA approved small molecules, along with safety switch mechanisms to eliminate replicon-encoded circuits in healthy cells. We will deliver our therapeutic circuits into mouse tumors in vivo and monitor anticancer immune responses, using systems biology principles to analyze the resulting response of multi-cell networks in the tumor. A key goal of these studies will be to design RNA circuits which drive transfected tumor/immune cells to act in trans on surrounding cells in the microenvironment, to achieve a tumor-wide change in the tumor milieu without the need for successful circuit delivery to every cell in the tumor. Results from this project will provide a ne RNA-based toolkit for engineering mammalian cell function, demonstrate the utility of these approaches in vivo, and provide a framework for reprogramming tumors that overcomes limitations of traditional chemotherapy and targeted drug treatments.