Improving Brain Recovery Through Glycoengineering

通过糖工程改善大脑恢复

• 批准号: 10666616
• 负责人: Xiaofeng Jia
• 金额: $ 48.23万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10666616
• 关键词: Adhesions; Affinity; Animal Model; Animals; Apoptosis; Behavior assessment; Behavioral; Biochemical; Biological; Biological Process; Biology; Brain; Brain Injuries; Cadherins; Carbohydrates; Cell Adhesion; Cell Communication; Cell surface; Cells; Cellular biology; Central Nervous System Diseases; Chemical Structure; Chemicals; Chemistry; Complex; Development; Dose; Electrophysiology (science); Engineering; Event; Extracellular Matrix; Foundations; Generations; Glycoconjugates; Glycoengineering; Gold; Healthcare; Heart Arrest; Human; Implant; In Vitro; Intercept; Ischemia; Kinetics; Lead; Malignant Neoplasms; Mass Spectrum Analysis; Mediator; Metabolic; Metabolism; Methodology; Modeling; Monosaccharides; N-acetylmannosamine; Nature; Nerve Regeneration; Neurites; Neuronal Differentiation; Neurons; Neurophysiology - biologic function; Organism; Pathway interactions; Pharmaceutical Preparations; Physiological; Polysaccharides; Preclinical Testing; Proteins; Rattus; Recovery; Recovery of Function; Reporting; Rodent Model; Safety; Sialic Acids; Signal Transduction; Spatial Distribution; Stimulus; Structure-Activity Relationship; Sulfhydryl Compounds; Surface; Synaptic plasticity; Techniques; Testing; Therapeutic; Time; Tissues; Translations; Transplantation; WNT Signaling Pathway; Wnt proteins; Work; analog; blastomere structure; cell motility; clinical application; clinical translation; conventional therapy; cost effectiveness; covalent bond; design; drug candidate; embryo cell; extracellular; functional group; functional improvement; glycoproteomics; healing; immune cell infiltrate; improved; in vivo; injured; injury recovery; innovation; intercellular communication; migration; nerve stem cell; neural; neuroregulation; novel; receptor; response; scaffold; stem cell biology; stem cell differentiation; stem cell fate; stem cell therapy; technology platform; therapeutic candidate; translational approach

Project Summary This project is based on recent advances in metabolic glycoengineering (MGE), a technology platform where non-natural monosaccharides intercept the biosynthetic pathways for cell surface-displayed glycans. As a result, chemical functionalities not naturally found in carbohydrates are installed in the glycalyx, which can alter cell adhesion, receptor activity, and downstream events (e.g., apoptosis, differentiation, and motility). In previous work, we developed the N-acetylmannosamine (ManNAc) analog “Ac5ManNTGc” to install thiol groups into sialic acids in human embryonic cells and found that – when the cells were grown on a “high affinity” surface (e.g., gold, which forms coordinate covalent bonds with thiols) – Wnt signaling was upregulated in the absence of extracellular Wnt proteins and neuronal differentiation was induced. In vivo translation of this approach, however, was hindered by the requirement for a non-degradable gold scaffold. We recently overcame this impediment by designing new ManNAc analogs with thiols presented on longer linkers, which extends this functional group further away from the core monosccharide and increases analog potency. Critically, the new analogs provide pro-neurogenic activity in the absence of a scaffold thereby simplifying in vivo translation. This project will explore analog mechanism in hNSCs in Specific Aim 1; this aim will define how the chemical structure, kinetics, and dose of thiol-modified ManNAc analogs (along with chemically inert size-matched controls) modulate cellular glycans in Aim 1a; evaluate changes to cell adhesion and motility in Aim 1b, and evaluate the differentiation of human neural stem cells (hNSCs) in Aim 1c. Next, in Specific Aim 2, we will apply the optimized analog-treatment conditions to improve neural regeneration in a rat cardiac arrest (CA) model of brain injury by transplanting MGE-modified into injured animals. We will compare hNSCs treated with our new thiol-modified analogs with appropriate controls on functional recovery after CA by evaluating survival, adhesion, distribution, and migration of transplanted hNSCs in rat brain. In Specific Aim 3, we will evaluate biochemical (Wnt signaling and cadherin involvement) and cellular (tissue infiltrating immune cells) level mechanisms we propose contribute to the healing effects of MGE in brain injury recovery (in Aim 3a). Finally, in Aim 3b we will characterize cell-wide “glycosites” by mass spectrometry and use glycobioinformatics analyses to identify unknown biochemical mediators of MGE. Specifically, we anticipate identifying new mediators of the beneficial effects of MGE in the implanted hNSCs as well as trans-acting host proteins. We hypothesize that thio-analogs modulate hNSC fate through a complex combination of receptor- specific effects on cell signaling and adhesion providing a pleiotropic suite of healing effects that cannot be achieved through conventional therapies. Accordingly, our innovative approach opens a new avenue to improve stem cell therapy with our new thiol-based MGE technique.

项目摘要 该项目基于代谢糖工程(MGE)的最新进展，MGE是一个技术平台， 非天然单糖拦截了细胞表面显示的多糖的生物合成途径。作为一名 结果，碳水化合物中天然不存在的化学功能被安装在糖萼中，这可能会改变 细胞黏附、受体活性和下游事件(例如，细胞凋亡、分化和运动)。在……里面 在之前的工作中，我们开发了N-乙酰甘露糖胺(ManNAc)类似物“Ac5ManNTGc”来安装硫醇 在人类胚胎细胞中形成唾液酸群并发现--当细胞生长在“高亲和力”上时 表面(例如，金，它与硫醇形成配位共价键)-Wnt信号在 细胞外Wnt蛋白缺失，诱导神经元分化。这一基因的体内翻译 然而，由于需要一个不可降解的黄金脚手架，这一方法受到了阻碍。我们最近 通过设计新的ManNAc类似物，使硫醇出现在较长的连接子上，克服了这一障碍 使该官能团进一步远离核心单糖，并增加模拟效价。 关键的是，新的类似物在没有支架的情况下提供了促神经生成活性，从而简化了 活体翻译。该项目将在具体目标1中探索hNSCs中的模拟机制；该目标将定义 硫醇修饰的ManNAc类似物的化学结构、动力学和剂量(以及化学惰性 大小匹配的对照)调节目标1a中的细胞糖链；评估细胞黏附和运动的变化 目的1b，并评价人神经干细胞(HNSCs)在Aim 1c中的分化情况。接下来，针对具体目标 2，我们将应用优化的模拟治疗条件来促进大鼠心脏神经再生 MGE基因修饰的脑损伤动物移植停滞(CA)模型我们将比较hNSC 用我们的新的硫醇修饰的类似物治疗，并适当控制CA后的功能恢复 评估移植的hNSCs在大鼠脑内的存活、黏附、分布和迁移。在具体目标3中， 我们将评估生化(Wnt信号和钙粘附素参与)和细胞(组织浸润性免疫 我们提出的细胞水平机制有助于MGE在脑损伤恢复中的修复作用(在AIM中 3A)。最后，在目标3b中，我们将通过质谱学表征细胞范围内的“糖点”，并使用 糖生物信息学分析以确定MGE的未知生化介体。具体来说，我们预计 在植入的hNSCs和反式作用宿主中识别MGE有益效应的新介体 蛋白质。我们假设硫代类似物通过一种复杂的受体组合来调节hNSC的命运- 对细胞信号和黏附的特定影响提供了一系列不能 通过传统疗法来实现。因此，我们的创新方法开辟了一条新的途径 使用我们新的基于硫醇的MGE技术改进干细胞治疗。