Precision monitoring of kidney transplants via single-cell and single-molecule sequencing

项目概要/摘要。 在美国，每年有超过15，000名患者接受挽救生命的肾脏移植。然而，尽管如此， 急性和慢性排斥反应引起的并发症经常发生，并限制了肾移植的寿命。 虽然临床医生努力仔细监测移植患者，但诊断选择仍然有限。诊断 肾移植中的排斥反应需要侵入性穿刺活检，并且感染的诊断具有挑战性 因为感染测试主要限于一次测试一种病原体。该提案概述了 全新的精确医学方法来监测肾移植。我们将发明并应用基因组 具有单细胞和单分子分辨率的分析，以剖析复杂的分子和细胞异质性 与重要的免疫学和感染性移植后并发症相关。组学分析将 对从肾移植受者队列中收集的700多份尿液样本进行了检测，可通过 与Manikkam Suthanthiran博士合作。在第一项研究中，我们将实现高通量单细胞 从肾移植受者尿液中分离的细胞测序，以了解，预测和诊断 肾移植并发症我们将研究与急性胰腺炎相关的单细胞基因表达谱， 缺血和再灌注损伤引起的细胞排斥、感染和移植物炎症。这项研究将采取 这是单细胞测序的最新进展的优势，其减轻了成本、规模和容易性的限制。第二、 我们将发明和应用非侵入性测量细胞类型和组织类型的特异性损伤， 通过分析尿无细胞DNA（cfDNA）检测肾脏。大量游离DNA的小片段 （cfDNA）存在于尿液中，是死细胞的碎片。我们将应用两种类型的精密测量 cfDNA的表观遗传改变具有高度的细胞、组织和器官类型特异性：胞嘧啶DNA甲基化 和DNA结合因子的全基因组占有率。这些测量将使得能够量化细胞， 尿cfDNA来源的组织类型，并将提供有关肾损伤相关的详细信息 移植并发症第三，我们将进行尿cfDNA的宏基因组学分析，以分析尿cfDNA的分布。 与尿路感染、多瘤病毒肾病和急性排斥反应相关的微生物组。我们将测试 利用这些测量来预测、理解和诊断泌尿系统的病毒和细菌感染， 道。微生物序列的鉴定并不能提供关于微生物生命周期的功能信息。 检测到的有机体为了从cfDNA的测量中获得功能性理解，我们将开发和 实施工具来描绘循环微生物组的结构。这些研究的成功实施 将使研究移植后并发症的生物学具有前所未有的分辨率，并将导致 新的，非侵入性的液体活检，以监测移植肾的健康。

Project Summary/Abstract. More than 15,000 patients receive lifesaving kidney transplants in the US every year. Nevertheless, complications due to acute and chronic rejection occur frequently and limit the lifespan of kidney transplants. While clinicians strive to monitor transplant patients carefully, diagnostic options remain limited. Diagnosis of rejection in kidney transplantation requires an invasive needle biopsy, and diagnosis of infections is challenging because tests of infection are predominantly limited to testing one pathogen at a time. This proposal outlines radically new precision-medicine approaches to kidney transplant monitoring. We will invent and apply genomic assays with single cell and single molecule resolution to dissect the complex molecular and cellular heterogeneity associated with important immunological and infectious post-transplant complications. Omics analyses will be performed on 700+ urine samples collected from a cohort of kidney transplant recipients, available through collaboration with Dr. Manikkam Suthanthiran. In a first study, we will implement high-throughput single-cell sequencing of cells isolated from the urine of kidney transplant recipients to understand, predict, and diagnose kidney transplant complications. We will investigate the single cell gene expression profiles associated with acute cellular rejection, infection and graft inflammation caused by ischemic and reperfusion injury. This study will take advantage of recent advances in single-cell sequencing that alleviate limitations of cost, scale and ease. Second, we will invent and apply noninvasive measurements of cell-type and tissue-type specific injury in transplanted kidneys via analyses of urinary cell-free DNA (cfDNA). A large number of small fragments of cell-free DNA (cfDNA) are present in urine that are the debris of dead cells. We will apply precision measurements of two types of epigenetic alterations of cfDNA that are highly cell-, tissue- and organ-type specific: cytosine DNA methylation and genome-wide occupancy of DNA-binding factors. These measurements will enable quantifying the cell and tissue types of origin of urinary cfDNA and will provide detailed information about injury associated with kidney transplant complications. Third, we will perform metagenomics analyses of urinary cfDNA to profile the urinary microbiome associated with urinary tract infection, polyomavirus nephropathy and acute rejection. We will test the utility of these measurements to predict, understand and diagnose viral and bacterial infections of the urinary tract. Identification of microbial sequences does not provide functional information about the life-cycle of the detected organism. To gain a functional understanding from measurements of cfDNA, we will develop and implement tools to profile the structure of the circulating microbiome. Successful implementation of these studies will enable studying the biology of post-transplant complications with unprecedented resolution and will lead to novel, noninvasive liquid biopsies to monitor the health of transplanted kidneys.