Decoding the Tissue of Origin of Cellular Damage from Cell-free DNA in Liquid Biopsies

摘要 液体活检应用正在迅速成为一种微创方法，用于收集系统范围内的 用于生理和疾病相关变化的基因组监测的代表性分析物。垂死细胞释放 DNA片段进入循环，称为无细胞DNA（cfDNA）。解码cfDNA的细胞起源 随着时间的推移，可以揭示改变的细胞贡献，反映了纵向组织损伤的动态变化。 问题研究在这里，我将集中讨论肝移植后细胞和组织稳态的变化， cfDNA分析细胞类型特异性甲基化模式将用于追踪cfDNA的细胞起源。 分子。此外，实体器官移植将与供体器官分开的DNA组放入体内 允许通过基因分型来识别和验证来自同种异体移植物的cfDNA分子 使用供体来源的SNP。在这个建议中，我的目标是跟踪细胞损伤后的变化组成， 肝移植，并利用这些信息来改善移植物功能障碍的诊断和管理（目标1）。 在移植过程中，组织驻留免疫细胞沿着着供体器官同时转移 组织.我将使用免疫细胞特异性DNA甲基化模式和供体SNP分析， 区分移植后和移植期间宿主和供体组织驻留免疫细胞的变化 免疫抑制治疗（目的2）。 拟议的研究将评估一种创新的方法，以深入了解宿主细胞的反应， 供体器官细胞，加上宿主和供体免疫细胞相对于不同的移植结果。一系列的证明- 原则性研究概述了使用肝移植作为一种理想的设置， 在特定的时间点，细胞内稳态将被诱导改变到一系列不同的基因组， 同种异体移植物中的细胞以及宿主。除了移植结果分析，cfDNA方法 根据这一建议建立的，可以扩展到确定细胞对组织损伤的贡献， 任何设置。细胞类型特异性甲基化模式是通用的标记，可用于追踪受损的 cfDNA的细胞来源，而不管损伤的原因。肝脏中的细胞损伤可能是由于靶向 治疗、化疗、免疫抑制或其他干预，原发性肝脏的开始或复发 在COVID-19大流行期间观察到的恶性肿瘤、癌症转移性播种或器官损伤。我们提出 将从不同类型的损伤中观察到不同的细胞cfDNA特征。此外，还有一个未解决的问题， 需要在开发新治疗方法的过程中深入了解组织损伤， 不良事件与疗效相关的细胞基础。这一提议的全球影响将是 将循环中的cfDNA与其细胞起源联系起来，从而揭示病理生理学的驱动因素。

ABSTRACT Liquid biopsy applications are rapidly emerging as a minimally invasive approach to collect system-wide representative analytes for genomic monitoring of physiologic and disease-related changes. Dying cells release fragmented DNA into the circulation, referred to as cell-free DNA (cfDNA). Decoding the cellular origins of cfDNA over time can reveal altered cellular contributions reflective of dynamic changes to tissue damages in longitudinal studies. Here, I will focus on the changes in cellular and tissue homeostasis post liver transplant using molecular analyses of cfDNA. Cell-type specific methylation patterns will be used to trace the cellular origins of cfDNA molecules. In addition, solid organ transplant place a separate DNA set with the donor organ into the body of the host, allowing cfDNA molecules from the allograft to be identified and validated through genotyping using donor-derived SNPs. In this proposal, I aim to track the changing composition of cellular damage post- liver transplant and use this information to improve diagnosis and management of graft dysfunction (Aim 1). During transplant there is simultaneous transfer of tissue-resident immune cells along with the donor organ tissue. I will use immune cell-specific DNA methylation patterns together with the donor SNP analysis to distinguish the host and donor tissue-resident immune cell changes after transplant and during immunosuppressive treatment (Aim 2). The proposed research will evaluate an innovative approach to gain insights into the reaction of host cells, donor organ cells, plus host and donor-immune cells relative to different transplant outcomes. A series of proof- of-principle studies are outlined using liver transplantation as an ideal setup that introduces an organ with a distinct genome at a specific timepoint where there will be induced changes in cell homeostasis to a range of cells in the allografts as well as the host. Beyond the transplant outcome analysis, the cfDNA approach established under this proposal can be expanded to determine the cellular contributions to tissue damages in any setting. Cell type-specific methylation patterns are universal markers that can be used to trace the damaged cell origin of cfDNA irrespective of the cause of damage. Cellular damage in the liver can be due to targeted therapy, chemotherapy, immunosuppression or other interventions, initiation or recurrence of primary liver malignancy, cancer metastatic seeding or organ damage observed during the COVID-19 pandemic. We propose that distinct cellular cfDNA signatures will be observed from different types of injury. Also, there is an unmet need to gain insights into tissue damage during the development of new treatments and understand the cellular basis of adverse events relative to therapeutic efficacy. The global impact of this proposal will be to link cfDNAs in the circulation to their cellular origins and thus reveal drivers of pathophysiology.