Development of a "Cell Splicing" Technology Platform

开发“细胞拼接”技术平台

• 批准号: 10578742
• 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 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10578742
• 关键词: Adherence; Adhesions; Affect; Autoimmunity; Behavior; Biological Assay; Blood Platelets; Bulla; 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; Macrophage; Mediating; Membrane; Membrane Fusion; Methods; Mitochondria; Monitor; Morphology; Mus; Nature; Nuclear; Organelles; Parents; Phagocytosis; Pharmaceutical Preparations; Play; Process; Proliferating; Protocols documentation; RNA Splicing; Research Project Grants; Research Proposals; Structure; Subcellular Fractions; System; T-Lymphocyte; Techniques; Temperature; Testing; Therapeutic; Therapeutic Intervention; Time; Variant; Vesicle; Wild Type Mouse; Work; cancer therapy; cell behavior; cell type; cytokine; density; improved; in vitro Assay; in vitro testing; in vivo; in vivo Model; interest; migration; novel therapeutics; nuclear transfer; perforin; preservation; reconstitution; response; synthetic biology; technology platform; tissue injury; tissue repair; 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.

项目概要： 一般科学界已经区分了不同的层或亚细胞部分（即膜与细胞）。 细胞质与细胞核）以及子成分细胞器/机械（例如线粒体、溶酶体等） 为了研究和更好地了解细胞功能。这项开拓者研究计划旨在发现如何 我们可能会重新调整这些组成部分的用途，目前主要强调核转让或交换作为其一部分 创造基于细胞的疗法的新合成生物学方法。这样的努力将带来发展 大规模扩展的介入治疗工具箱，具有广泛的潜在下游 生物医学中的应用（即癌症、遗传和传染病、自身免疫和组织疾病的治疗） 损伤和修复）。这将通过以下方式实现： 1) 生成有效地从巨噬细胞和 T 细胞中分离细胞核以融合成的方法 去核红细胞和血小板。首先将使用药物和药物来优化核分离方法 密度离心诱导细胞起泡和分级分离以分离细胞核与细胞质成分 含有囊泡，分别称为核质体和细胞质。核质体将源自先天 免疫巨噬细胞和适应性免疫 T 细胞，然后融合（与 PEG）到自然去核的红细胞中 随着时间的推移，将监测血小板和衍生细胞构建体的活力和功能。 2) 制定储存、冷冻和解冻要求，以维持细胞来源的活力 细胞质和核质体以及融合构建体。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协议温度动力学以存储（短与长） 术语）以及解冻细胞或其成分并保留结构和功能（图 1，中）。 3) 表征巨噬细胞和 T 细胞来源的细胞质，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。先前的摘除研究表明 改变了细胞行为，因此不仅对研究核交换感兴趣，而且对发生的情况也感兴趣 去核细胞。此外，将尝试用新鲜和冷冻核质体进行核交换 和细胞质成分，所有融合构建体都经过形态/活力、增殖、细胞因子测试 源自供体细胞或不同于供体细胞的表达和行为。这种方法还将使我们能够 确定如何将结构作为更大的即插即用系统的一部分进行调整。 4) 在体外功能测定和体内癌症治疗模型中测试新构建体。这 该策略将提供一个平台来创建与巨噬细胞等功能活动相关的新细胞行为 相关的粘附和吞噬作用，以及 T 细胞介导的穿孔素/颗粒酶细胞溶解作用。所以， 构建体将在体外进行粘附、迁移、共培养（细胞溶解）测定以及抗肿瘤试验 小鼠体内活性。