Ultrasound-Controlled Immunotherapy for Targeted Treatment of Solid Tumors

项目摘要 合成生物学的进步使得越来越多的基于细胞的诊断技术的发展成为可能。 和治疗工具。然而，在体内控制这些细胞是困难的，目前的方法来沟通， 而工程化的细胞要么具有较差的时空分辨率，要么需要侵入性操作。为了 基于细胞疗法安全性和有效性的提高， 必需的.温度是一种独特的通信信号，因为它可以以毫米精度进行调制 使用聚焦超声（FUS）非侵入性地在组织内进行。另外，细胞已经进化出了 通过热休克启动子（HSP）来感知温度的变化。这些遗传因子无处不在 在生物体中，提供了一个平台来开发将在FUS加热时激活的遗传电路。结合 FUS提供的空间控制与细胞工程赋予热敏性将使我们能够创造 FUS治疗后将在特定位置激活的热响应细胞。 一个可以从空间控制的基因表达中获益的快速发展的领域是基于细胞的癌症 免疫疗法免疫疗法最近已成为一种有前途的新型癌症疗法， 在血液恶性肿瘤中的变革性结果。然而，基于工程细胞的免疫疗法， 由于CAR T细胞和免疫调节剂如细胞因子必须克服之前的重大挑战， 变得更加广泛地适用于实体肿瘤。在最近的临床试验中，CAR-T细胞已经攻击了健康的 如果它们的靶向抗原不是肿瘤限制性的，则它们会在组织中产生毒性，导致大量的外周毒性和死亡。系统性 细胞因子疗法也可引起危及生命的副作用，例如血管渗漏综合征。两种类型的 免疫治疗可受益于空间控制的治疗性表达。 该项目的总体目标是在初级T细胞中开发热休克蛋白驱动的电路， 通过FUS递送至活性治疗基因表达。最初的实验将集中在培养T细胞， 通过瞬时释放细胞因子来响应整体加热，以增强自分泌中的CAR性能 在肿瘤中特异性地激活永久CAR表达。我们将通过开发 HSP驱动的电路具有药物诱导的反式激活因子或Cre蛋白酶。在验证了这些电路之后 在体外，我们将使用鼠SKBR 3肿瘤模型和小鼠肿瘤模型测试它们在体内FUS治疗后的性能。 抗HER 2 CAR。该模型将使我们能够开发和展示强大的，FUS激活的性能 具有两个不同有效载荷输出的原代T细胞。这种方法代表了一种新的组合， 细胞工程和治疗性超声，以时空控制基于细胞的免疫疗法， 直接应用于实体瘤治疗。这项技术也具有进一步发展的巨大潜力 以及适应其他细胞类型，这可能有助于基础科学和转化研究。

Project Summary Advances in synthetic biology have enabled the development of increasing numbers of cell-based diagnostic and therapeutic tools. However, controlling these cells in vivo is difficult and current methods to communicate with engineered cells either suffer from poor spatiotemporal resolution or require invasive operations. In order to improve safety and efficacy of cell-based therapies, robust methods to control gene expression in vivo are required. Temperature is a unique communication signal as it can be modulated with millimeter precision deep within tissues non-invasively using focused ultrasound (FUS). In addition, cells have already evolved the ability to sense changes in temperature through heat shock promoters (HSPs). These genetic elements are ubiquitous across organisms, providing a platform to develop genetic circuits that will activate upon FUS heating. Combining the spatial control offered by FUS with cellular engineering to confer thermal sensitivity will allow us to create thermally responsive cells that will activate in specific locations following FUS treatment. One rapidly growing field that could benefit from spatially controlled gene expression is cell-based cancer immunotherapy. Immunotherapy has recently emerged as a promising new class of cancer therapies with transformative results in hematological malignancies. However, engineered cell-based immunotherapies such as CAR T-cells and immunomodulatory agents such as cytokines must overcome significant challenges before becoming more widely applicable for solid tumors. In recent clinical trials, CAR T-cells have attacked healthy tissues if their targeted antigen is not tumor limited, resulting in massive peripheral toxicity and death. Systemic cytokine therapy can also cause life-threatening adverse effects such as vascular leak syndrome. Both types of immunotherapy could benefit from spatially controlled therapeutic expression. This project’s overall goal is to develop HSP-driven circuits in primary T-cells that will allow thermal stimuli delivered by FUS to active therapeutic genes expression. Initial experiments will focus on developing T-cells in vitro that will respond to bulk heating by transiently releasing cytokine to boost CAR performance in an autocrine fashion or activating permanent CAR expression specifically in the tumor. We will accomplish this by developing HSP driven circuits that feature drug induced transactivators or Cre Recombinase. After validating these circuits in vitro, we will test their performance upon FUS treatment in vivo using a murine SKBR3 tumor model and an anti-HER2 CAR. This model will allow us to develop and demonstrate the performance of robust, FUS-activated primary T-cells with two distinct payload outputs. This proposed approach represents a novel combination of cellular engineering and therapeutic ultrasound to spatiotemporally control cell-based immunotherapies with direct application to solid tumor treatment. This technology also has substantial potential for further development and adaption to other cell types which may facilitate both basic science and translational research.