Development of a "Cell Splicing" Technology Platform

项目总结： 一般的科学界已经将不同的层或亚细胞组分分开(即，膜与 细胞质和细胞核)以及亚成分细胞器/机械(如线粒体、溶酶体等)。 以便研究和更好地了解细胞的功能。这份开拓者的研究提案试图发现 我们可以改变这些组成部分的用途，目前的主要重点是核转让或交换，作为一部分 一种新的合成生物学方法来创造基于细胞的疗法。这样的努力将导致发展 大规模扩展的介入性治疗工具箱，具有广泛的潜在下游 在生物医学(即癌症、遗传和传染病、自身免疫和组织治疗)中的应用 受伤和修复)。这将通过以下方式实现： 1)创造有效地从巨噬细胞和T细胞中分离细胞核以融合到 去核的红细胞和血小板。核分离方法将首先使用药物和 密度离心法诱导细胞起泡和分离，以分离细胞核和细胞质成分 含有囊泡，分别称为核质和细胞质。有核细胞将来自先天的 免疫巨噬细胞和获得性免疫T细胞，然后(与聚乙二醇)融合成自然去核的红细胞 和血小板，以及衍生的细胞结构将随着时间的推移而监测其活性和功能。 2)制定储存、冷冻和解冻要求，以保持细胞来源的活性 细胞质和有核体，以及融合结构。这将通过探索不同的冷冻介质来实现 两个商店的类型、成分化学浓度或更改的协议温度动力学(短与长 术语)以及具有保存结构和功能的解冻细胞或其组件(图1，中间)。 3)鉴定巨噬细胞和T细胞来源的细胞质，以及下列新的变异细胞 去核巨噬细胞与T细胞体之间的核交换。先前的摘除研究表明 改变了细胞的行为，因此，研究核交换以及发生了什么不仅是有意义的 去核细胞。此外，还将尝试用新鲜的和冷冻的有核体进行核交换。 和细胞质组件，所有融合构建体都经过了形态/活性、增殖、细胞因子的测试 表达，以及源自或不同于供体细胞的行为。这种方法还将使我们能够 确定如何将构造作为更大的即插即用系统的一部分进行调整。 4)在体外功能分析和体内治疗癌症模型中测试新构建物。这 战略将提供一个平台，创造与功能活动相关的新细胞行为，如巨噬细胞- 相关的黏附和吞噬作用，以及T细胞介导的穿孔素/颗粒酶细胞溶解。因此， 构建物将在体外进行黏附、迁移和共培养(细胞溶解)试验以及抗肿瘤试验。 在小鼠体内活动。

Project Summary: The general scientific community already separates different layers or subcellular fractions (i.e., membrane vs. cytoplasm vs. nucleus) as well as subcomponent organelles/machinery (such as mitochondria, lysosomes, etc.) in order to study and better understand cell function. This Trailblazer research proposal seeks to discover how we might repurpose such components, with major emphasis presently on nuclear transfer or exchange as part of a new synthetic biology approach in creating cell-based therapies. Such efforts will lead to the development of a massively expanded toolbox of interventional therapies with a wide array of potential downstream applications in biomedicine (i.e., treatments for cancer, genetic and infectious disease, autoimmunity, and tissue injury and repair). This will be achieved through the following: 1) Generate methods to efficiently isolate nuclei from macrophage and T cells for fusion into enucleated red blood cells and platelets. Methods for nuclear isolation will first be optimized using drug and density centrifugation-induced cellular blebbing and fractionation to isolate nuclei- vs. cytoplasmic component- containing vesicles, called karyoplasts and cytoplasts, respectfully. Karyoplasts will be derived from innate immune macrophage and adaptive immune T cells, and then fused (with PEG) into naturally enucleated RBCs and platelets, and derived cell constructs will be monitored for viability and function over time. 2) Develop storage, freezing, and thawing requirements to maintain viability of cell-derived cytoplasts and karyoplasts, and fusion constructs. This will be done by exploring different freezing media types, constituent chemical concentrations, or altered protocol temperature kinetics to both store (short vs. long- term) as well as thaw cells or their components with preserved structure and function (Figure 1, middle). 3) Characterize macrophage- & T cell-derived cytoplasts, as well as new variant cells following nuclear exchange between enucleated macrophage and T cell bodies. Prior enucleation studies show modified cell behavior, therefore it is not only of interest to investigate nuclear exchange but also what happens to enucleated cells. In addition, nuclear exchange will be attempted with both fresh as well as frozen karyoplast and cytoplast components, with all fusion constructs tested for morphology/viability, proliferation, cytokine expression, and behaviors either derived or distinct from donor cells. This approach will also allow us to determine how constructs may be tunable as part of a larger plug and play system. 4) Test new constructs in functional assays in vitro and in a therapeutic cancer model in vivo. This strategy will provide a platform to create new cell behaviors related to functional activities like macrophage- related adherence and phagocytosis, as well as T cell-mediated perforin/granzyme cytolysis. Therefore, constructs will be tested in vitro in adhesion, migration, and co-culture (cytolysis) assays as well as for anti-tumor activity in vivo in mice.