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

项目名称：高分辨率三维原位空间基因表达谱技术 人脑标本 项目说明 此第一阶段项目的总体目标是应用扩展排序，a 具有前所未有的空间分辨率的全基因组原位转录图谱技术 在3D中，以人脑组织为动力进行脑部疾病研究和治疗开发。 神经退行性疾病，如阿尔茨海默病，影响着超过11%的人口 65岁以上，导致老年人⅓死亡，每年造成数千亿美元的损失。 然而，还没有疾病修改疗法被批准上市。有能力获得 数据和验证直接在人类样本中的发现对我们的能力至关重要 描述并理解大脑紊乱的特征。空间分辨转录组学，帮助 科学家使用荧光显微镜了解不同的细胞是如何组织的 成像在表征自然组织中不同类型的细胞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动物大脑的薄层切片。 它们受到光学衍射限制的分辨率的限制，限制了精确定义 以3D形式组织的各种单元类型。来自人类的组织和那些 神经退行性疾病是由蛋白质聚集体引起的高度自发荧光。 (如淀粉样斑块)、脂褐素颗粒和致密血管。最近发表于 科学，扩展测序(ExSeq)是第一个原位全基因组三维空间基因 表情分析技术。它在3D中提供前所未有的成像分辨率，使用Thick 小鼠脑切片。这使得能够清楚地定义突触连接和定位基因 单细胞和亚细胞精度的转录本，这是 大多数研究人员使用的常规荧光共聚焦显微镜。为了使 ExSeq适用于人体研究并可用于商业用途，我们发现并测试了一种新的 一组方法，使我们能够优化人体样本的ExSeq，并改进 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 提高分析管道的稳定性。我们还获得了一个广泛的 各种珍贵的人脑组织，帮助我们测试不同的样品制备条件 并验证我们的方法。 对于这个第一阶段的项目，我们将为人脑组织开发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和 分析管道，可容纳从不同的实验和 实验室环境。最后，我们将处理和分析一组人类正常和 阿尔茨海默氏病的脑组织，并对照已发表的数据和之前的数据验证结果 研究。在强大的科学基础和出版物的支持下，我们正在 汇聚了协议优化和排序技术方面的广泛专业知识 ExSeq和对阿尔茨海默氏症和神经退行性疾病病理的深入了解 它是基础科学和治疗研究和开发的有效工具。

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.