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.

项目总结 EpiCypher正在与Jessica Tyler博士(衰老、DNA修复和表观遗传学专家)合作，以 开发一流的CUT&RUSSNTM(靶下切割并使用单链核酸酶释放) 单链DNA作图技术在早期发病机制研究中的应用 阿尔茨海默病(AD)的干预措施。基因组DNA的双链构象(DsDNA) 对维持基因组稳定至关重要。单链DNA在许多细胞过程中形成，包括转录和 DNA损伤的处理，并被单链DNA结合蛋白(SSB)(例如RPA，RAD51)快速隔离 和BRCA1/BRCA2)来保护和促进任何所需的修复。广告是最常见的形式 神经变性，早期发病/神经细胞死亡，部分原因是DNA损伤的积累 作为缺陷修复机制的结果(特别是同源重组[HR]，这是严重的 依赖于单链DNA信号通路)。单链DNA和单链B-单链DNA检测定位方法的改进 伴随DNA损伤修复的复合体将极大地提高我们对这些损伤修复失败的理解 通路有助于AD，并可能揭示新的药物靶点和生物标记物。然而，研究的工具 缺乏与单链DNA相关的信号。我们方法的第一个创新是开发了一部小说 免疫系留方法，其中：1)针对单链DNA相关特征(例如SSB)的抗体用于局部 2)下一步，核酸酶被激活以 选择性地切割附近的单链DNA而不是双链DNA；以及3)收集切割的片段并对其进行测序 产生精确的单链DNA目标定位轮廓。蛋白A/G(PAg)与单链DNA融合的研究进展 特异性核酸酶是一项关键的创新，因为它能够最终鉴定与任何 本地化因素。我们方法的第二个创新是开发核小体尖峰控制 含有ssDNA或dsDNA，将用于：1)确认核酸酶特异性；和2)使 疾病/对照样本的定量比较-/+最终药物治疗。这一阶段的目标是 项目是开发CUT和RUSSN工作流程(目标1)，并展示其绘制SSB-ssDNA图谱的能力 因此，能够对阿尔茨海默病模型中的单链DNA修复途径进行新的研究(目标2)。在第二阶段， 我们将扩展Cut&RUSSN平台以增加染色质功能(例如SSB或组蛋白PTM)及其 相关的细胞机制(如转录、R-环、DNA复制)。此外，我们还将发展强大的 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 自动化化验以实现大规模临床研究。在第二阶段结束时，我们将推出CUT&RUSSN 试剂盒和化验服务，将向研究人员、药物开发商和临床医生营销，以加快 广告药物发现。