High-resolution 3D in situ Spatial Gene Expression Profiling Technology for Human Brain Specimens

人脑标本高分辨率3D原位空间基因表达谱分析技术

• 批准号: 10385072
• 负责人: Mahender Babu Dewal
• 金额: $ 50万
• 依托单位: EXPANSION TECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10385072
• 关键词: 3-Dimensional; Academia; Affect; Aging; Alzheimer' s Disease; Alzheimer' s disease brain; American; Amyloid; Animal Experiments; Animal Model; Animals; Area; Astrocytes; Basic Science; Biological; Biological Sciences; Brain; Brain Diseases; Cataloging; Cause of Death; Cells; Chemistry; Chromosome Mapping; Collagen; Cytoplasmic Granules; Data; Data Analytics; Databases; Detection; Development; Diagnosis; Digestion; Disease; Drug Targeting; Ensure; Erythrocytes; Excision; Fluorescence; Fluorescence Microscopy; Formaldehyde; Foundations; Gene Expression; Gene Expression Profiling; Genes; Homologous Gene; Human; Image; In Situ; Individual; Industry; Investigational Drugs; Knowledge; Laboratories; Lateral; Legal patent; Libraries; Lipofuscin; Marketing; Metastatic breast cancer; Methods; Microglia; Microscope; Microscopy; Mission; Modeling; Molecular; Mus; Names; Nature; Neurodegenerative Disorders; Neurons; Noise; Optics; PF4 Gene; Pathologic; Pathology; Patients; Phase; Population; Preparation; Process; Protocols documentation; Public Health; Publications; Publishing; RNA; Reaction Time; Reagent; Reproducibility; Research; Research Personnel; Resolution; Sampling; Science; Scientist; Senile Plaques; Sensitivity and Specificity; Signal Transduction; Specimen; Speed; Standardization; Synapses; System; Technology; Temperature; Testing; Therapeutic; Therapeutic Human Experimentation; Thick; Thinness; Tissues; Transcript; Update; Visualization software; aged; analytical tool; animal tissue; base; behavior influence; biomarker discovery; brain research; brain tissue; cell type; clinical biomarkers; cost; data analysis pipeline; data pipeline; design; fluorescence imaging; gene panel; genome-wide; global health; high resolution imaging; human disease; human tissue; image processing; image registration; imaging system; improved; invention; microscopic imaging; new technology; protein aggregation; research and development; sample fixation; therapeutic development; tool; transcriptomics; usability; validation studies

Project Title: High-resolution 3D in situ Spatial Gene Expression Profiling Technology for Human Brain Specimens Project Description The overall aim of this Phase I project is to apply Expansion Sequencing, a genome-wide in situ transcriptomics profiling technology with unprecedented spatial resolution in 3D, to human brain tissues to empower brain disease research and therapeutic development. Neurodegenerative diseases, such as Alzheimer’s disease affects over 11% of the population aged above 65, causing ⅓ of death in seniors, and costs hundreds of billions of dollars a year. Yet, no disease modifying therapeutics have been approved for marketing. The ability to obtain data and validate discoveries directly in human samples is paramount to our ability to characterize and understand brain disorders. Spatially resolved transcriptomics, helps scientists understand how the different cells are organized, using fluorescence microscopy imaging has shown unmatched promise in characterizing different cell types in native tissue, change during development and aging, and how they influence behavior and disease. However, many of existing spatial technologies are limited to thin animal brain sections. They are bound by optical diffraction-limited resolution, restraining the ability to precisely define a large variety of cell types organized in 3D. Tissues from humans and those with neurodegenerative disease have high degree of autofluorescence caused by protein aggregates (such as Amyloid plaques), lipofuscin granules and dense vessels. Recently published in Science, Expansion Sequencing (ExSeq) is the first in situ genome-wide 3D spatial gene expression profiling technology. It provides unprecedented imaging resolution in 3D using thick mouse brain sections. This allows for clear definition of synapse junctions and mapping gene transcripts with single- and sub-cellular precision, which had not been possible with conventional fluorescence confocal microscopes used by most researchers. In order to make ExSeq suitable for human studies and commercially available, we identified and tested a new set of methods that will allow us to optimise ExSeq for human specimens, and improve sensitivity and specificity. We are building a set of analytical tools to help visualize, debug and improve the robustness of the analytics pipeline. We have also obtained access to a wide variety of precious human brain tissues, to help us test different sample preparation conditions and validate our methods. For this Phase I project, we will develop ExSeq protocol for human brain tissue characterisation with a proof-of-concept gene panel, and create a robust image processing and analytical pipeline that can accommodate images generated from different experimental and laboratory settings. Finally, we will process and analyse a set of human normal and Alzheimer’s diseased brain tissues, and validate results against published data and prior research. Building upon a strong scientific foundation supported with publications, we are bringing together extensive expertise in protocol optimization and sequencing technology for ExSeq and deep knowledge of Alzheimer’s and neurodegenerative disease pathology to make it an impactful tool for both basic science and therapeutic research and development.

项目名称：高分辨率3D原位空间基因表达谱技术 人脑标本 项目描述 这个第一阶段项目的总体目标是应用扩展排序， 具有前所未有的空间分辨率的全基因组原位转录组学分析技术 在3D中，人类大脑组织，使大脑疾病的研究和治疗发展。 神经退行性疾病，如阿尔茨海默病，影响超过11%的人口 65岁以上的老年人，造成老年人死亡，每年花费数千亿美元。 然而，还没有疾病修饰治疗剂被批准上市。能够获得 直接在人体样本中进行数据采集和验证， 描述和理解大脑疾病。空间分辨转录组学，有助于 科学家们利用荧光显微镜了解不同细胞的组织方式， 成像在表征天然组织中的不同细胞类型方面显示出无与伦比的前景， 在发育和衰老过程中的变化，以及它们如何影响行为和疾病。 然而，许多现有的空间技术仅限于薄的动物脑切片。 它们受到光学衍射极限分辨率的限制，限制了精确定义 各种各样的细胞类型以3D的形式组织起来。从人类和那些 神经变性疾病具有由蛋白质聚集体引起高度自发荧光 (such淀粉样斑块）、脂褐质颗粒和致密血管。最近发表在 科学，扩展测序（ExSeq）是第一个原位全基因组3D空间基因 表达谱分析技术。它提供了前所未有的3D成像分辨率， 小鼠脑切片。这使得突触连接的清晰定义和基因定位成为可能 转录单和亚细胞精度，这是不可能的， 大多数研究人员使用的传统荧光共聚焦显微镜。为了使 ExSeq适合于人类研究并且可商购获得，我们鉴定并测试了一种新的 一套方法，使我们能够优化人类标本的ExSeq，并提高 敏感性和特异性。我们正在构建一套分析工具，以帮助可视化，调试和 提高分析管道的鲁棒性。我们还获得了一个广泛的 多种珍贵的人脑组织，帮助我们测试不同的样品制备条件 验证我们的方法 对于这个第一阶段项目，我们将开发用于人脑组织的ExSeq协议。 使用概念验证基因面板进行表征，并创建强大的图像处理， 分析管道，可以容纳从不同的实验和 实验室设置。最后，我们将处理和分析一组人类正常， 阿尔茨海默氏病的脑组织，并验证结果对已发表的数据和先前 research.建立在出版物支持的强大科学基础上，我们 汇集了广泛的专业知识，在协议优化和测序技术， ExSeq和阿尔茨海默氏症和神经退行性疾病病理学的深刻知识， 它是基础科学和治疗研究与开发的有效工具。