Development of a "Cell Splicing" Technology Platform

开发“细胞拼接”技术平台

• 批准号: 10426268
• 负责人: Joshua Charles Doloff
• 金额: $ 20.47万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-10 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10426268
• 关键词: Adherence; Adhesions; Affect; Autoimmunity; Behavior; Biological Assay; Blood Platelets; Cancer Model; Cell Line; Cell Nucleus; Cell Survival; Cell Therapy; Cell fusion; Cell physiology; Cells; Cellular Structures; Centrifugation; Chemicals; Coculture Techniques; Communicable Diseases; Communities; Cytochalasin B; Cytolysis; Cytoplasm; Development; Drug usage; Engineering; Erythrocytes; Evolution; Foundations; Fractionation; Freezing; Genetic Diseases; Genetic Recombination; Goals; Granzyme; Heritability; Human; Hybrids; Immune; Individual; Kinetics; Knock-out; Lipid Bilayers; Lysosomes; Mediating; Membrane; Membrane Fusion; Methods; Mitochondria; Monitor; Morphology; Mus; Nature; Nuclear; Organelles; Parents; Phagocytosis; Pharmaceutical Preparations; Play; Process; Protocols documentation; RNA Splicing; Research Project Grants; Research Proposals; Structure; Subcellular Fractions; System; T-Lymphocyte; Techniques; Technology; Temperature; Testing; Therapeutic; Therapeutic Intervention; Time; Variant; Vesicle; Wild Type Mouse; Work; base; cancer therapy; cell behavior; cell type; cytokine; density; improved; improved functioning; in vitro Assay; in vitro testing; in vivo; in vivo Model; injury and repair; innovation; interest; macrophage; migration; novel therapeutics; nuclear transfer; perforin; preservation; reconstitution; response; synthetic biology; tissue injury; tool; tumor

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.

项目摘要： 一般科学界已经将不同的层或亚细胞部分（即，膜与 细胞质与细胞核）以及亚组分细胞器/机器（如线粒体、溶酶体等）。 来研究和更好地理解细胞功能。这项开拓者研究计划旨在发现 我们可以重新利用这些部件，目前主要强调核转让或交换， 一种新的合成生物学方法来创造基于细胞的疗法。这些努力将导致发展 大规模扩展的介入治疗工具箱，具有广泛的潜在下游 在生物医学中的应用（即，治疗癌症、遗传和传染病、自身免疫和组织 损伤和修复）。这将通过以下方式实现： 1)产生有效地从巨噬细胞和T细胞分离细胞核以用于融合成 去核红细胞和血小板。核分离的方法将首先使用药物和 密度离心诱导的细胞起泡和分级以分离细胞核-与细胞质成分- 含有囊泡，分别称为核质体和胞质体。核质体将来自先天的 免疫巨噬细胞和适应性免疫T细胞，然后融合（与PEG）到天然去核RBC中 和血小板，以及衍生的细胞构建体将随时间监测存活力和功能。 2)制定储存、冷冻和解冻要求，以维持细胞源性 细胞质和核质，以及融合构建体。这将通过探索不同的冷冻介质来完成 类型、成分化学浓度或改变的协议温度动力学，以存储（短与长） 术语）以及解冻细胞或其结构和功能保留的组分（图1，中）。 3)表征巨噬细胞和T细胞衍生的胞质，以及以下新的变异细胞 去核巨噬细胞和T细胞体之间的核交换。先前的眼球摘除研究显示 改变细胞的行为，因此，它不仅是感兴趣的研究核交换，而且发生了什么 去核细胞此外，将尝试用新鲜和冷冻的核质体进行核交换 和细胞质组分，测试所有融合构建体的形态/活力、增殖、细胞因子 表达和行为来源于或不同于供体细胞。这种方法还将使我们能够 确定如何将构造作为较大的即插即用系统的一部分进行可调。 4)在体外功能测定和体内治疗性癌症模型中测试新构建体。这 战略将提供一个平台，创造新的细胞行为有关的功能活动，如巨噬细胞- 相关的粘附和吞噬作用，以及T细胞介导的穿孔素/颗粒酶细胞溶解。因此，我们认为， 构建体将在体外进行粘附、迁移和共培养（细胞溶解）试验以及抗肿瘤试验 小鼠体内活性。