Graft Engineering of Allogeneic Hematopoietic Stem Cell Products with Molecular Cascades

分子级联同种异体造血干细胞产品的移植工程

• 批准号: 9426101
• 负责人: Markus Y. Mapara
• 金额: $ 65.82万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-01 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9426101
• 关键词: Acute; Address; Adverse effects; Allogenic; Animal Disease Models; Antibodies; Antigens; B-Lymphocytes; Benchmarking; Blood Component Removal; CD3 Antigens; CD34 gene; CD4 Positive T Lymphocytes; Cell Separation; Cell Therapy; Cell physiology; Cell surface; Cells; Chronic; Clinic; Clinical; Clinical Trials; Color; Complex; Complex Mixtures; Complication; DNA; Development; Disease model; Elements; Engineering; Excision; Flow Cytometry; Fluorescence; Goals; Health; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Human; IL2RA gene; Immune; Immunomagnetic Separation; Immunotoxins; In Situ; Label; Magnetism; Mediating; Memory; Methods; Molecular; Mus; Natural Killer Cells; Oligonucleotides; Outcome; Patients; Population; Preparation; Prevention strategy; Procedures; Process; Property; Protocols documentation; Reaction; Regulatory T-Lymphocyte; Relapse; Signal Transduction; Stem cells; Structure; Surface; Testing; Time; Transplantation; antibody conjugate; aptamer; base; clinical application; clinically relevant; cost; graft function; graft vs host disease; humanized mouse; improved; in vivo; in vivo evaluation; mouse model; population based; prevent; targeted treatment; tool

Proposal Summary Subpopulations of hematopoietic cells with different functions are identified based on the presence or absence of cell surface markers. Certain subpopulations are hypothesized to mediate forms of graft-vs-host disease (GVHD) in allogeneic hematopoietic stem cell transplantations (aHSCT). The most advanced efforts to minimize these side effects, and to have reached clinics, rely on an extended three step immunomagnetic separation protocol with sequential positive selection (for protection of stem cells from elimination based on CD34 expression), negative selection (elimination of naïve TN-cells based on CD45RA expression), and subsequent return of positively selected stem cells to the graft that is then introduced into patients. The engineered graft, however, is also depleted of B-, NK-, and Treg-cells, which are essential for control of infectious complications, modulation of GVHD, and relapse control, thus impacting long-term survival after transplantation. Rapid and efficient single-step positive or negative selections that would minimize handling of cells out of their natural niches would also improve cell health likely leading to better clinical outcomes. Highl precision single-step procedures would also provide us with a powerful tool for rigorous and reliable studies of the impact of high quality preparations of specific subpopulations of cells in animal models of disease. We have recently developed molecular computing cascades based on oligonucleotides conjugated to targeting moieties (such as antibodies, their fragments, and aptamers). These are mixtures of targeting moieties that carry molecular computing elements and produce one outcome, a specific label on a single cell subpopulation, based on taking into account all targeting moieties. Thus, what would be, for example, a three-step separation protocol or a three-colored-fluorescence characterization in flow cytometry is condensed into a single step isolation procedure or a single color in flow cytometry. These computing mixtures of oligonucleotide conjugates with targeting moieties are thus uniquely suited for single-step `mix-and-separate' cell labelling protocols for magnetic separations. Our cascades can block isolation based on the presence of a cell surface marker or they can amplify signal coming from surface markers with low expression levels. In this project we will ask two principal questions: (1) What are the practical limitations, with metrics being yield, purity, and health of cells, of molecular computing cascades when applied to isolation of cell-subpopulations for clinical applications? And, (2) What are the minimal subpopulation of cells that we have to either preserve or eliminate to minimize induction of various forms of GVHD. In three Aims we will address translational and mechanistic questions regarding graft engineering and the induction of GVHD using increasingly complex protocols. We will characterize the impact of cell populations with different levels of markers for naïve, central memory, and Treg cells in a mouse model of GVHD, while at the same time assessing translational potential of increasingly complex molecular computing cascades, in comparison to standard methods.

提案摘要 基于存在或不存在造血干细胞，鉴定具有不同功能的造血细胞亚群。 细胞表面标志物。假设某些亚群介导移植物抗宿主病的形式 异基因造血干细胞移植（aHSCT）中的移植物抗宿主病（GVHD）。最先进的努力， 最大限度地减少这些副作用，并已达到诊所，依靠扩展的三步免疫磁性 具有顺序阳性选择的分离方案（用于保护干细胞免于基于 阴性选择（基于CD 45 RA表达消除幼稚TN细胞），和 随后将阳性选择的干细胞返回移植物，然后将其引入患者体内。的 然而，工程化的移植物也耗尽了B-、NK-和Treg-细胞，这些细胞对于控制肿瘤生长是必需的。 感染并发症、GVHD调节和复发控制，从而影响移植后的长期生存率。 移植快速有效的单步阳性或阴性选择，最大限度地减少 细胞从它们的自然生态位中出来也会改善细胞健康，可能导致更好的临床结果。海格尔 精确的单步程序也将为我们提供一个强大的工具， 特定细胞亚群的高质量制剂在疾病动物模型中的影响。 我们最近开发了分子计算级联的基础上结合到靶向寡核苷酸 部分（如抗体、其片段和适体）。这些是靶向部分的混合物， 携带分子计算元件并产生一种结果，在单个细胞亚群上的特定标记， 基于考虑所有靶向部分。因此，例如，三步分离 将流式细胞术中的三色荧光表征浓缩为单个步骤 分离程序或流式细胞术中的单一颜色。这些计算寡核苷酸的混合物 因此，具有靶向部分的缀合物独特地适合于单步“混合-分离”细胞标记 磁性分离的协议。我们的级联可以基于细胞表面的存在来阻止隔离 标记物，或者它们可以放大来自具有低表达水平的表面标记物的信号。 在这个项目中，我们将问两个主要问题：（1）什么是实际的限制，与指标产量， 当应用于细胞亚群的分离时， 临床应用？（2）我们必须保存或保存的最小细胞亚群是什么？ 消除，以尽量减少各种形式的GVHD的诱导。在三个目标中，我们将解决翻译和 关于移植物工程和GVHD诱导的机制问题， 协议.我们将描述具有不同水平标志物的细胞群对幼稚、中枢神经系统和神经系统的影响。 在小鼠GVHD模型中，同时评估 越来越复杂的分子计算级联，与标准方法相比。