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

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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.