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

摘要 Triangle Biotechnology正在开发ChromExtract™，这是一种低成本的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。这些技术是表观基因组研究和绘制的核心。 组蛋白修饰、增强子和染色质状态，使表观基因组效应的系统研究成为可能 对细胞身份、发育、谱系特化和疾病的影响。虽然染色质谱分析已经 二十多年来，存在着几个瓶颈，阻碍了表观遗传分析的产生， 来自FFPE组织的一致和可靠的数据，最明显的是与当前的FFPE组织相关的质量和产量差。 染色质提取方法。这是一个相当大的问题，因为大多数转化研究和临床 诊断使用FFPE组织样本。此外，与FFPE组织的相容性提供了获得生物相容性的途径。 大量的档案样本库，可用于进一步表征 兴趣考虑到该领域的现状，快速、高产、高效地提取 来自FFPE组织的染色质，从而提高染色质质量和样本类型的标准化 将对研究染色质可及性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分析。目前还没有商用解决方案， 分析FFPE组织样品中的染色质可及性。这种类型的分析采用非目标 系统查询与基因表达相关的染色质开放区域的方法， 研究人员广泛了解染色质状态的变化如何影响疾病进展， 特定组蛋白标记或转录因子分布的改变。Triangle的ChromExtract提供了一个 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 使用低成本的全氟化碳（PFC）纳米液滴基试剂， 在任何市售超声仪中从FFPE组织中提取染色质。在第一阶段SBIR项目中， Triangle Bio将：1）在实验室中展示纳米液滴制剂增强的空化作用和增加的弹性。 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， 甲醛辅助的调节元件分离（FAIRE）这项工作将建立可行性， 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，产生的结果是严格的，可重复的，有效的和成本效益。这 在未来的第二阶段应用中，将通过其他下游基于NGS的工作流程进行进一步验证。

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.