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与Jessica Tyler博士（衰老，DNA维修和表观遗传学专家）合作， 开发切割和russNTM（靶标下裂解，并使用单链核酸酶释放），这是第一类 单链DNA（ssDNA）映射技术，用于研究早期发病机理和可能 阿尔茨海默氏病的干预措施（AD）。基因组DNA（dsDNA）的双链构象为 维持基因组稳定性至关重要。在许多细胞过程中形成ssDNA，包括转录和 DNA病变的加工​​，并通过ssDNA结合蛋白（SSB）迅速隔离（例如RPA，Rad51 和BRCA1/BRCA2）保护和促进任何必要的维修。广告是最常见的形式 神经变性，早期发病机理 /神经元细胞死亡部分归因于DNA损伤的积累 由于修复机制有缺陷（尤其是同源重组[HR]，这是严重的 依赖于ssDNA信号通路）。改进的检测和映射ssDNA和SSB-SSDNA的方法 适应DNA损伤修复的复合物将大大提高我们对这些失败的理解 途径有助于AD，并有可能揭示新的药物靶标和生物标志物。但是，学习的工具 缺乏与ssDNA相关的信号传导。我们方法的第一个创新是小说的发展 免疫方法，其中：1）使用与ssDNA相关特征（例如SSB）的抗体用于局部 系链囊核中的ssDNA特异性核酸酶在透化核中的染色质； 2）接下来，将核酸酶激活为 有选择地清除附近的ssDNA，而不是dsdna； 3）收集分裂的片段并将 产生精确的ssDNA目标定位曲线。蛋白A/G（PAG）的发展与ssDNA- 特定的核酸酶是关键创新，因为它可以确定与任何相关的ssDNA的确定性 本地化因素。我们方法的第二个创新是核探针尖峰控制的发展 包含ssDNA或dsDNA，将使用：1）确认核酸酶特异性； 2）启用 疾病 /对照样本中的定​​量比较 - /+事件药物治疗。这个阶段的目标 项目将开发剪切工作流（AIM 1），并证明其映射SSB-SSDNA的能力 细胞中的复合物，从而实现了AD模型中ssDNA修复途径的新研究（AIM 2）。在第二阶段， 我们将将切割和RUSSN平台扩展到其他染色质功能（例如SSB或Hisstone PTMS）及其 相关的细胞机制（例如转录，R环，DNA复制）。此外，我们将发展强大 广泛研究的AD模型和人类验尸大脑的协议，包括低细胞输入应用和 测定自动化以实现大规模临床研究。在第二阶段结束时，我们将推出一个切割和RUSSN Beta-kit和Assay服务将向研究人员，药物开发人员和临床医生推销，以加速 AD药物发现。