High-resolution genomic mapping of ssDNA and associated proteins for Alzheimer's disease research

用于阿尔茨海默病研究的 ssDNA 和相关蛋白的高分辨率基因组图谱

• 批准号: 10382044
• 负责人: Michael-Christopher Keogh
• 金额: $ 50万
• 依托单位: EPICYPHER, 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/10382044
• 关键词: Aging; Alzheimer' s Disease; Alzheimer' s disease model; Alzheimer' s disease pathology; Antibodies; Automation; Autopsy; BRCA1 gene; BRCA2 gene; Bar Codes; Benchmarking; Binding Proteins; Biological Assay; Biological Markers; Brain; Cause of Death; Cell Nucleus; Cell model; Cell physiology; Cells; ChIP-seq; Chromatin; Clinical Research; Complex; DNA; DNA Binding; DNA Repair Pathway; DNA mapping; Development; Disease Progression; Epigenetic Process; Failure; GTP-Binding Protein alpha Subunits, Gs; Genetic Transcription; Genome Stability; Genomic Segment; Genomic approach; Genomics; Goals; Histones; Human; Hybrids; Intervention; Invaded; Lesion; Mammalian Cell; Maps; Measures; Mediating; Methods; Micrococcal Nuclease; Molecular Conformation; Nerve Degeneration; Neurons; Nucleosomes; Pathogenesis; Pathway interactions; Performance; Pharmaceutical Preparations; Pharmacotherapy; Phase; Play; Post-Translational Protein Processing; Process; Prognostic Marker; Proteins; Protocols documentation; RAD52 gene; RNA; Research; Research Personnel; Resolution; Role; SS DNA BP; Sampling; Services; Signal Pathway; Signal Transduction; Single-Stranded DNA; Specificity; Stretching; Technology; Yeasts; biomarker discovery; clinical application; cost; disorder control; drug development; drug discovery; ds-DNA; epigenomics; homologous recombination; improved; innovation; neuron loss; new therapeutic target; novel; nuclease; recombinational repair; repaired; research and development; targeted biomarker; tool

PROJECT SUMMARY EpiCypher is collaborating with Dr. Jessica Tyler (an expert in aging, DNA repair and epigenetics), to develop CUT&RUssNTM (Cleavage Under Targets and Release Using single-stranded Nuclease), a first-in-class single-stranded DNA (ssDNA) mapping technology for research into the early pathogenesis of and possible interventions for Alzheimer’s Disease (AD). The double-stranded conformation of genomic DNA (dsDNA) is essential to maintain genome stability. ssDNA forms during many cellular processes, including transcription and the processing of DNA lesions, and is rapidly sequestered by ssDNA binding proteins (SSBs) (e.g. RPA, RAD51 and BRCA1/BRCA2) to protect and facilitate any needed repair. AD is the most common form of neurodegeneration, with early pathogenesis / neuronal cell death due in part to the accumulation of DNA damage as a consequence of defective repair mechanisms (particularly homologous recombination [HR], which is heavily reliant on ssDNA signaling pathways). Improved methods for detecting and mapping ssDNA and SSB-ssDNA complexes that accompany DNA damage repair would greatly improve our understanding of how failure of these pathways contributes to AD, and potentially reveal novel drug targets and biomarkers. However, tools to study ssDNA-related signaling are lacking. The first innovation of our approach is the development of a novel immunotethering approach, wherein: 1) an antibody to an ssDNA-associated feature (e.g. SSB) is used to locally tether an ssDNA-specific nuclease to chromatin in permeabilized nuclei; 2) next, the nuclease is activated to selectively cleave nearby ssDNA and not dsDNA; and 3) cleaved fragments are collected and sequenced to yield a precise ssDNA target localization profile. The development of protein A/G (pAG) fused to an ssDNA- specific nuclease is a key innovation, as it enables the definitive identification of ssDNA associated with any localizing factor. A second innovation of our approach is the development of nucleosome spike-in controls containing either ssDNA or dsDNA, which will be used: 1) to confirm nuclease specificity; and 2) to enable quantitative comparisons in disease / control samples -/+ eventual drug treatment. The goals of this Phase I project are to develop the CUT&RUssN workflow (Aim 1) and demonstrate its ability to map SSB-ssDNA complexes in cells, thus enabling the novel study of ssDNA repair pathways in AD models (Aim 2). In Phase II, we will expand the CUT&RUssN platform to additional chromatin features (e.g. SSBs or histone PTMs) and their associated cellular mechanisms (e.g. transcription, R-loops, DNA replication). In addition, we will develop robust protocols for widely studied AD models and human post-mortem brains, including low cell input applications and assay automation to enable large-scale clinical studies. At the end of Phase II, we will launch a CUT&RUssN beta-kit and assay services, which will be marketed to researchers, drug developers, and clinicians to accelerate AD drug discovery.

项目摘要 EpiCypher正在与Jessica泰勒博士（衰老，DNA修复和表观遗传学专家）合作， 开发CUT&RUssNTM（使用单链核酸酶进行靶下切割和释放）， 单链DNA（ssDNA）作图技术用于研究早期发病机制和可能的 阿尔茨海默病（AD）的治疗。基因组DNA（dsDNA）的双链构象是 对维持基因组稳定至关重要。ssDNA在许多细胞过程中形成，包括转录和 DNA损伤的处理，并被ssDNA结合蛋白（SSB）（例如RPA、RAD 51）迅速隔离 和BRCA 1/BRCA 2），以保护和促进任何需要的修复。AD是最常见的 神经变性，伴早期发病机制/部分由于DNA损伤累积导致的神经元细胞死亡 由于有缺陷的修复机制（特别是同源重组[HR]， 依赖于ssDNA信号传导途径）。检测和定位ssDNA和SSB-ssDNA的改进方法 伴随DNA损伤修复的复合物将极大地提高我们对这些修复失败的理解。 研究表明，这些信号通路有助于AD，并可能揭示新的药物靶点和生物标志物。然而，研究工具 ssDNA相关信号缺乏。我们方法的第一个创新是开发一种小说 1）使用针对ssDNA相关特征（例如SSB）的抗体局部地 将ssDNA特异性核酸酶系在透性化的核中的染色质上; 2）接着，激活核酸酶， 选择性地切割附近的ssDNA而不是dsDNA;和3）收集切割的片段并测序， 产生精确的ssDNA靶定位谱。蛋白A/G（pAG）融合到ssDNA的发展- 特异性核酸酶是一个关键的创新，因为它能够确定性地鉴定与任何 局部化因子我们方法的第二个创新是核小体加标控制的发展 包含ssDNA或dsDNA，其将用于：1）确认核酸酶特异性;和2）使 疾病/对照样品的定量比较-/+最终药物治疗。第一阶段的目标 该项目旨在开发CUT&RUssN工作流程（目标1）并展示其映射SSB-ssDNA的能力 复合物在细胞中，从而使新的研究ssDNA修复途径在AD模型（目的2）。在第二阶段， 我们将把CUT&RUssN平台扩展到其他染色质特征（例如SSB或组蛋白PTM）及其 相关的细胞机制（例如转录、R环、DNA复制）。此外，我们还将大力发展 广泛研究的AD模型和人类死后大脑的方案，包括低细胞输入应用， 检测自动化，支持大规模临床研究。在第二阶段结束时，我们将推出CUT&RUssN β试剂盒和检测服务，将向研究人员，药物开发人员和临床医生销售，以加速 AD药物发现。