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

项目摘要/摘要。 在美国，每年有超过1.5万名患者接受挽救生命的肾脏移植手术。不过， 急性和慢性排斥反应引起的并发症频繁发生，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 肾移植的排斥反应需要有侵入性的针吸活组织检查，感染的诊断具有挑战性。 因为对感染的检测主要限于一次检测一种病原体。这份提案概述了 肾移植监测的全新精确医学方法。我们将发明并应用基因组 用单细胞和单分子分辨分析复杂的分子和细胞异质性 与移植后重要的免疫学和感染性并发症有关。组学分析将是 对从肾移植受者队列中收集的700份尿样进行了检测，这些样本可以通过 与Manikkam Suthan Thiran博士合作。在第一项研究中，我们将实现高通量的单细胞 肾移植受者尿液中分离细胞的测序以了解、预测和诊断 肾移植并发症。我们将研究与急性白血病相关的单细胞基因表达谱。 缺血再灌注损伤引起的细胞排斥、感染和移植物炎症。这项研究将需要 利用单细胞测序的最新进展，缓解了成本、规模和简单性的限制。第二, 我们将发明并应用非侵入性测量移植中的细胞型和组织型特异性损伤。 通过分析尿液中的游离DNA(CfDNA)检测肾脏。大量的无细胞DNA小片段 (CfDNA)存在于尿液中，是死亡细胞的碎片。我们将应用两种类型的精密测量 高度细胞、组织和器官类型特异性的cfDNA表观遗传学改变：胞嘧啶DNA甲基化 以及DNA结合因子在全基因组中的占有率。这些测量将使量化细胞和 尿cfDNA来源的组织类型，并将提供与肾脏相关的损伤的详细信息 移植并发症。第三，我们将对尿液cfdna进行元基因组学分析，以描述尿液的情况。 与尿路感染、多瘤病毒肾病和急性排斥反应相关的微生物群。我们将测试 这些测量在预测、了解和诊断尿路病毒和细菌感染方面的作用 一条小路。对微生物序列的鉴定不提供有关微生物生命周期的功能信息 检测到的有机体。为了从cfDNA的测量中获得功能理解，我们将开发和 使用工具来描述循环微生物群的结构。成功实施这些研究 将能够以前所未有的分辨率研究移植后并发症的生物学，并将导致 用于监测移植肾脏健康的新型非侵入性液体活组织检查。

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.