A Novel Class of Synthetic Receptors to Empower the Age of mRNA Therapies

摘要 冠状病毒疫苗接种活动强调了信使核糖核酸介导的递送作为一种新的 治疗方式。一种特殊的应用是过继细胞疗法，其中免疫细胞配备了 治疗疾病的新功能，例如在T细胞中表达嵌合抗原受体(CARS)以去除癌症 和其他不良细胞。与基于DNA的免疫细胞工程相比，信使核糖核酸有几个方面 作为传递媒介的优势，特别是其卓越的安全性，因为它消除了随机 插入宿主基因组并引起突变，及其短暂的半衰期可以减轻长期的不利影响 由于基因工程细胞功能的持久性。工程技术不是从病人身上提取细胞，而是 在体外，然后重新诱导它们，研究人员甚至直接传递了编码汽车的mRNA和 在体内创造了具有功能的CAR T细胞。这很有吸引力，因为它有可能成为领养细胞 公众可获得的治疗，其物流几乎像制造、分销、 和注射疫苗，相比之下，昂贵的体外工程过程将仅限于 享有特权的少数人。 尽管信使核糖核酸介导的过继细胞疗法具有巨大的潜力，但仍然迫切需要 提高目标精确度的工具。以汽车为目标的单一表面标记非常罕见 明确地确定一个目标细胞群体。因此，“对准目标/远离癌症”的杀戮是 CAR T细胞疗法对抗癌症，同样的担忧也适用于切除其他细胞的情况， 如心肌梗死或衰老细胞中活跃的成纤维细胞。一种优雅的解决方案是合成受体，大多数 特别是合成凹槽，它检测到第二个标记并响应快车，有效地形成和 逻辑上，只有当合成受体和CAR的输入都存在时，靶细胞才会被杀死。然而， 据我们所知，所有现有的合成模块化、可编程受体都在DNA水平上工作，并且 因此与信使核糖核酸介导的传递不相容。 在这里，我们设计并论证了一种一流的合成模块化受体的可行性 在RNA水平上。它将配体诱导的二聚化事件转化为任意输出蛋白的表达。 通过广泛的计算模拟和实验优化，我们将扩大投入/产出 该受体的谱系，建立其设计原则，使其能够在单个转录本上编码，并通过 并朝着提高去除活性成纤维细胞治疗心肌梗死的精确度迈出了第一步。 这种新型受体的影响超出了过继细胞疗法的范畴。例如，它们可以促进基本的 通过记录细胞(例如神经元)暴露于特定信号(例如多巴胺)的研究。他们将受益于 各种其他生物医学应用，从表达抗原或细胞因子对细胞外的反应 提高疫苗效力的线索，为组织工程产生新的感觉和反应能力。

Abstract The COVID vaccination campaigns have highlighted the promise of mRNA-mediated delivery as a novel therapeutic modality. One particular application is adoptive cell therapy, where immune cells are equipped with novel functions to treat diseases, e.g., expressing chimeric antigen receptors (CARs) in T cells to ablate cancer and other undesirable cells. Compared to DNA-based engineering of immune cells, mRNA has several advantages as a delivery vector, especially its superior safety profile, because it eliminates the risk of randomly inserting into the host genome and causing mutations, and its short half-life mitigates long-term adverse effects due to the persistence of the engineered cellular function. Instead of extracting cells from the patient, engineering them ex vivo, and then reinducing them, researchers have even directly delivered CAR-encoding mRNAs and created functional CAR T cells in vivo. This is appealing because it has the potential to make adoptive cell therapy accessible to the general public, its logistics almost as straightforward as manufacturing, distributing, and administering vaccine shots, in contrast to the costly ex vivo engineering process that will be limited to the privileged few. Despite the great potential of mRNA-mediated adoptive cell therapy, there remains a critical need for tools that enhance targeting precision. It is very rare for a single surface marker, targeted by CAR, to unambiguously identify one target cell population. Therefore, “on-target/off-caner” killing is a major concern for CAR T cell therapies against cancer, and the same concern also applies to scenarios of ablating other cells, such as active fibroblasts in heart infarction or senescent cells. One elegant solution is synthetic receptors, most notably synthetic Notch, that detect a second marker and express CAR in response, effectively forming AND logic, where target cells are only killed when both inputs to the synthetic receptor and CAR are present. However, to our knowledge, all existing synthetic modular, programmable receptors operate at the DNA level, and are therefore incompatible with mRNA-mediated delivery. Here we design and demonstrate the feasibility of a first-in-class synthetic modular receptor that operate at the RNA level. It converts ligand-induced dimerization events into the expression of arbitrary output proteins. Through extensive computational simulation and experimental optimization, we will expand the input/output repertoire of this receptor, establish its design principle, enable its encoding on single transcripts and delivery by mRNA, and take first steps towards improving the precision of ablating active fibroblasts to treat heart infarction. The impact of such novel receptors is beyond adoptive cell therapy. For example, they can facilitate basic research by recording cells’ (e.g., neurons) exposure to specific signals (e.g., dopamine). They will benefit a variety of other biomedical applications too, from expressing antigens or cytokines in response to extracellular cues to enhance vaccine efficacy, to generating novel sense-and-respond capabilities for tissue engineering.