Development of nanodroplet enhanced ultrasonic cavitation technologyto enable the study of chromatin accessibility in FFPE tissues

开发纳米液滴增强超声空化技术以实现 FFPE 组织中染色质可及性的研究

• 批准号: 10699112
• 负责人: David F Allison
• 金额: $ 33.93万
• 依托单位: TRIANGLE BIOTECHNOLOGY, INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-15 至 2024-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10699112
• 关键词: Acceleration; Affect; Benchmarking; Biological Assay; Biotechnology; Buffers; Cell Lineage; Cells; Chromatin; Cytolysis; Data; Detergents; Development; Diagnostic; Digestion; Disease; Disease Progression; Enhancers; Ensure; Enzymes; Epigenetic Process; Event; Extracellular Matrix; FAIRE sequencing; Fluorocarbons; Formalin; Formulation; Future; Gene Expression; Genetic Transcription; Genome; Histones; Industry Standard; Investigation; Laboratories; Life; Maps; Medical Research; Methods; Modeling; Pain; Paraffin Embedding; Pathology; Performance; Phase; Process; Protocols documentation; Publishing; Reagent; Reproducibility; Reproducibility of Results; Research; Research Personnel; Salts; Sampling; Small Business Innovation Research Grant; Sonication; Specific qualifier value; Specimen; Standardization; Techniques; Technology; Therapeutic; Time; Tissue Embedding; Tissue Sample; Translational Research; Ultrasonics; Validation; Work; chromatin immunoprecipitation; clinical diagnostics; commercialization; cost; cost comparison; cost effective; cost effectiveness; crosslink; epigenetic profiling; epigenomics; flexibility; histone modification; human disease; improved; instrument; interest; molecular subtypes; nanoDroplet; novel; prevent; promote resilience; tissue processing; transcription factor

Abstract Triangle Biotechnology is developing ChromExtract™, a low-cost, nanodroplet-based sonication reagent for chromatin extraction from formalin-fixed paraffin-embedded (FFPE) tissues for downstream chromatin profiling and chromatin accessibility analyses. These techniques are central to epigenomic research and mapping of histone modifications, enhancers, and chromatin states, enabling systematic investigation of epigenomic effects on cell identity, development, lineage specification, and disease. While chromatin profiling analyses have been used for over two decades, several bottlenecks exist that have prevented epigenetic profiling from yielding consistent and reliable data from FFPE tissues, most notably the poor quality and yield associated with current chromatin extraction methods. This is a considerable problem, as most translational research and clinical diagnostics employ FFPE tissue specimens. Moreover, compatibility with FFPE tissues provides access to a massive bank of archival samples that can be used to further characterize molecular subtypes for pathologies of interest. Given the current state of the field, a rapid, high-yield, and high-efficiency method for extraction of chromatin from FFPE tissue that results in improved chromatin quality and standardization across sample types would have a significant impact on the field and potential applications for studying chromatin accessibility and other epigenetic assays in medical research. There are currently no commercially available solutions for analyzing chromatin accessibility in FFPE tissue samples. This type of analysis employs a non-targeted approach to systematically query open regions of chromatin associated with gene expression, allowing researchers to broadly understand how changes in chromatin state affect disease progression independent of alterations in specific histone marks or transcription factor distribution. Triangle’s ChromExtract provides a workflow that employs an extracellular matrix (ECM)-digesting enzyme cocktail followed by enhanced sonication using a low-cost, perfluorocarbon (PFC) nanodroplet-based reagent for high-quality, high-yield, and consistent chromatin extraction from FFPE tissue in any commercially available sonicator. In this Phase I SBIR project, Triangle Bio will: 1) Demonstrate enhanced cavitation and increased resilience of nanodroplet formulation in FFPE-based lysis buffers and 2) Optimize and validate the FFPE tissue chromatin extraction workflow for Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) seq. This work will establish the feasibility of commercializing ChromExtract by demonstrating the ability to enable NGS-based analysis of chromatin extracted from FFPE samples, producing results that are rigorous, reproducible, efficient, and cost effective. This will be followed in a future Phase II application by further validation with other downstream NGS-based workflows.

抽象的 Triangle Biotechnology 正在开发 ChromExtract™，这是一种低成本、基于纳米液滴的超声处理试剂，用于 从福尔马林固定石蜡包埋 (FFPE) 组织中提取染色质，用于下游染色质分析 和染色质可及性分析是表观基因组研究和绘图的核心。 组蛋白修饰、增强子和染色质状态，从而能够系统地研究表观基因组效应 细胞身份、发育、谱系规范和疾病。 使用了二十多年，存在一些阻碍表观遗传分析产生的瓶颈 来自 FFPE 组织的一致且可靠的数据，最显着的是与当前相关的质量和产量较差 与大多数转化研究和临床一样，这是一个相当大的问题。 诊断采用 FFPE 组织样本，此外，与 FFPE 组织的兼容性提供了获取样本的途径。 大量档案样本可用于进一步表征病理学的分子亚型 鉴于该领域的现状，寻找一种快速、高产、高效的提取方法。 来自 FFPE 组织的染色质，可提高染色质质量和跨样本类型的标准化 将对研究染色质可及性和研究的领域和潜在应用产生重大影响 医学研究中的其他表观遗传学检测目前还没有商业可用的解决方案。 分析 FFPE 组织样本中的染色质可及性 此类分析采用非靶向方法。 系统地查询与基因表达相关的染色质开放区域的方法，允许 研究人员广泛了解染色质状态的变化如何独立影响疾病进展 Triangle 的 ChromExtract 提供了特定组蛋白标记或转录因子分布的改变。 采用细胞外基质 (ECM) 消化酶混合物，然后进行增强超声处理的工作流程 使用低成本、基于全氟化碳 (PFC) 纳米液滴的试剂可获得高质量、高产率和一致的结果 在任何市售超声波仪中从 FFPE 组织中提取染色质 在第一阶段 SBIR 项目中， Triangle Bio 将： 1) 展示纳米液滴配方增强的空化作用和增强的弹性 基于 FFPE 的裂解缓冲液和 2) 优化和验证 FFPE 组织染色质提取工作流程 甲醛辅助调节元件分离 (FAIRE) seq. 通过展示基于 NGS 的染色质分析能力将 ChromExtract 商业化 从 FFPE 样品中提取，产生严格、可重复、高效且具有成本效益的结果。 随后将在未来的第二阶段应用中通过其他下游基于 NGS 的工作流程进行进一步验证。