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.

项目概要 EpiCypher 正在与 Jessica Tyler 博士（衰老、DNA 修复和表观遗传学专家）合作， 开发 CUT&RUssNTM（目标下切割并使用单链核酸酶释放），这是一流的 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) 的干预措施 基因组 DNA (dsDNA) 的双链构象是 在许多细胞过程中，包括转录和转录过程中，对于维持基因组稳定性至关重要。 DNA 损伤的处理，并被 ssDNA 结合蛋白 (SSB)（例如 RPA、RAD51）快速隔离 和 BRCA1/BRCA2）以保护和促进任何所需的修复，是最常见的 AD 形式。 神经变性，早期发病/神经元细胞死亡，部分原因是 DNA 损伤的积累 由于有缺陷的修复机制（特别是同源重组 [HR]，这严重影响 改进的 ssDNA 和 SSB-ssDNA 检测和作图方法 伴随 DNA 损伤修复的复合物将极大地提高我们对这些缺陷如何失效的理解 然而，这些途径有助于 AD，并可能揭示新的药物靶点和生物标志物。 我们的方法的第一个创新是开发一种新颖的方法。 免疫束缚方法：1) 使用针对 ssDNA 相关特征（例如 SSB）的抗体来局部检测 将 ssDNA 特异性核酸酶连接至透化细胞核中的染色质；2) 接下来，激活核酸酶 选择性地切割附近的 ssDNA 而不是 dsDNA；以及 3) 收集切割的片段并进行测序 产生精确的 ssDNA 靶定位图谱 融合到 ssDNA 的蛋白 A/G (pAG) 的开发。 特异性核酸酶是一项关键创新，因为它能够明确识别与任何物质相关的单链DNA。 我们方法的第二个创新是核小体掺入控制的发展。 包含 ssDNA 或 dsDNA，将用于：1) 确认核酸酶特异性；2) 启用 疾病/对照样本的定量比较 -/+ 最终药物治疗 这一阶段的目标。 项目将开发 CUT&RUssN 工作流程（目标 1）并展示其绘制 SSB-ssDNA 的能力 细胞中的复合物，从而能够对 AD 模型中的 ssDNA 修复途径进行新的研究（目标 2）。 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 此外，我们将开发强大的相关细胞机制（例如转录、R 环、DNA 复制）。 用于广泛研究的 AD 模型和人类死后大脑的协议，包括低细胞输入应用和 检测自动化以实现大规模临床研究 在第二阶段结束时，我们将启动 CUT&RUssN。 beta 试剂盒和检测服务，将向研究人员、药物开发商和忠实拥护者推销，以加速 AD药物发现。