Development of a "Cell Splicing" Technology Platform

开发“细胞拼接”技术平台

基本信息

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
项目类别：
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

项目摘要

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分数分开（即膜VS。细胞质与核）以及子组合细胞器/机械（例如线粒体，溶酶体等）为了学习并更好地了解细胞功能。这项开拓者的研究建议旨在发现我们可能会重新利用此类组件，目前重点是核转移或交换为一部分一种新的合成生物学方法来创建基于细胞的疗法。这样的努力将导致发展大规模扩展的介入疗法工具箱，并具有各种潜在的下游电位在生物医学（即癌症，遗传和传染病，自身免疫和组织的治疗方法）中的应用伤害和维修）。这将通过以下以下方面实现： 1）生成有效分离核核与巨噬细胞和T细胞融合到的方法联核红细胞和血小板。首先使用药物优化核隔离方法，并密度离心诱导的细胞泄漏和分级分离，以分离核 - 细胞质成分 - 恭喜地包含称为核成形物和细胞质的囊泡。核成形物将源自先天免疫巨噬细胞和适应性免疫T细胞，然后融合（带钉）自然浓缩的RBC 随着时间的推移，将监视血小板以及衍生的细胞构建体以保持生存力和功能。 2）制定存储，冷冻和解冻要求，以维持细胞衍生的生存能力细胞质和核成形物和融合构建体。这将通过探索不同的冷冻媒体来完成类型，组成化学浓度或对两种储存的方案温度动力学改变（短与长期）术语）以及融化细胞或其具有保留结构和功能的组件（图1，中间）。 3）表征巨噬细胞和细胞衍生的细胞体以及随后的新变体细胞联核巨噬细胞和T细胞体之间的核交换。事先摘除研究表明修改后的细胞行为，因此研究核交换不仅有兴趣，而且还会发生什么诱发细胞。此外，将尝试使用新鲜和冷冻的核成形体进行核交换和细胞体成分，所有融合构建体测试了形态/生存力，增殖，细胞因子表达和行为衍生或与供体细胞不同。这种方法也将使我们能够确定构造如何作为较大的插头和播放系统的一部分进行调谐。 4）在体外和体内的治疗性癌症模型中测试功能测定中的新结构。这策略将提供一个平台，以创建与巨噬细胞等功能活动相关的新细胞行为相关的依从性和吞噬作用，以及T细胞介导的穿孔/颗粒胞质溶液。所以，构建体将在粘附，迁移和共培养（细胞溶解）测定中进行体外测试以及抗肿瘤小鼠体内活性。