Genome Transplant Dynamics: non-invasive sequencing-based diagnosis of rejection

描述（由申请人提供）：我们的目标是开发和测试一种新的供体特异性基因组方法，用于实体器官移植后排斥反应和移植物功能障碍的非侵入性早期诊断，并解决两个目标主题领域：1）应用基因组学和其他高通量技术; 2）将基础科学发现转化为新的更好的治疗方法。器官移植挽救了终末期器官衰竭患者的生命，但在许多情况下，移植的器官被受体排斥，导致危及生命的情况。先前开发移植物排斥的非侵入性标志物的尝试集中在免疫特异性免疫应答上，因此在灵敏度和特异性方面具有固有的局限性，特别是对于区分排斥与感染。我们的新方法是第一个专注于急性排斥反应的供体特异性标志物。我们将使用高通量下一代测序来监测受体血流中无细胞供体DNA与受体DNA的比例，作为排斥反应的标志。这种方法是通过器官移植也是有效的基因组移植的事实来实现的，并且通过监测对供体基因组特异性的单核苷酸多态性，可以测量移植器官的相对健康。依据/假设：在排斥过程中，供体细胞的凋亡将供体DNA释放到受体循环中;该DNA可以与受体DNA区分开，并使用高通量测序技术进行定量;供体DNA水平的动态变化将预测（a）急性排斥;（B）移植物功能障碍;和（c）慢性排斥。初步研究：使用来自心脏移植受者的库存血液样本，我们发现供体DNA水平在急性排斥反应期间升高，但在没有排斥反应的情况下保持在稳定的低水平。目的1：建立一种无创性检测供者DNA的方法。我们将开发和测试：（1）下一代DNA测序方法，以进行供体DNA载量的低成本、非侵入性分析;（2）生物信息学算法，其最大化区分供体和受体DNA的灵敏度;（3）区分供体和受体的定义的单核苷酸多态性（SNP）组的灵敏度。目标2：评价供体DNA监测在心脏移植后临床事件（急性排斥、慢性排斥和移植物功能障碍）检测中的实用性。我们将对110名连续心脏移植受者进行前瞻性队列研究，在子宫内膜异位症活检（EMB）过程中和之间采集血液样本（产生1，516对EMB/血液），以确定供体：受体DNA比例是否可以在早期阶段和移植物功能障碍发生之前检测排斥反应。目标3：在肺移植中测试这种方法，并确定供体：受体DNA比例是否可以区分由于排斥反应和肺部感染引起的移植物功能障碍。如果能够实现，这些具体目标将为验证研究供体特异性、基于基因组的方法提供基础，以非侵入性、早期检测实体器官移植后的排斥反应。 公共卫生相关性：器官移植挽救了终末期器官衰竭患者的生命，但在许多情况下，移植的器官被受体排斥，导致危及生命的情况。我们建议开发和测试一种新的和创新的方法，以非侵入性的，早期诊断排斥反应和移植物功能障碍后，心脏和肺移植。通过简单地监测移植受者的血液，以确定接受移植后随访期间出现的供体基因组水平，我们希望建立一种诊断工具，该工具有可能每年节省数亿美元的医疗保健费用，并降低患者的发病率和死亡率。

DESCRIPTION (provided by applicant): Our goal is to develop and test a novel donor-specific, genomic approach to the non-invasive, early diagnosis of rejection and graft dysfunction after solid organ transplantation, and addresses two targeted thematic areas: 1) Applying Genomics and Other High Throughput Technologies; 2) Translating Basic Science Discoveries into New and Better Treatments. Organ transplantation saves the lives of patients with end-stage organ failure, yet in many cases, the transplanted organ is rejected by the recipient, causing a life-threatening situation. Previous attempts to develop a non-invasive marker of graft rejection have focused on recipient-specific immune responses, and thus have inherent limitations in sensitivity and specificity, especially for distinguishing rejection from infection. Our novel approach is the first to focus on a donor-specific marker of acute rejection. We will use high throughput next generation sequencing to monitor the proportion of cell-free donor DNA to recipient DNA, in the recipient's blood stream as a marker of rejection. This approach is enabled by the fact that an organ transplant is also effectively a genome transplant, and by monitoring single nucleotide polymorphisms that are specific to the donor's genome one can measure the relative health of the transplanted organ. RATIONALE/HYPOTHESIS: during rejection, apoptosis of donor cells releases donor DNA into the recipient circulation; this DNA can be distinguished from recipient DNA and quantified using high-throughput sequencing techniques; and dynamic changes in donor DNA levels will predict (a) acute rejection; (b) graft dysfunction; and (c) chronic rejection. PRELIMINARY STUDIES: using banked blood samples from heart transplant recipients, we showed that donor DNA levels rise during episodes of acute rejection but remain at stable, low levels in the absence of rejection. AIM 1: Develop a donor DNA monitoring approach for the non-invasive detection of allograft rejection. We will develop and test: (1) next generation DNA sequencing methods to perform low cost, noninvasive analysis of donor DNA load; (2) bioinformatic algorithms that maximize the sensitivity to discriminate donor and recipient DNA; (3) sensitivity of a defined single nucleotide polymorphism (SNP) panel that differentiates donor from recipient. AIM 2: Evaluate the utility of donor DNA monitoring for detection of clinical events (acute rejection, chronic rejection, and graft dysfunction) after heart transplantation. We will conduct a prospective cohort study of 110 consecutive heart transplant recipients, collecting blood samples during and between endomyocardial biopsy (EMB) procedures (yielding 1,516-paired EMB/blood) to determine whether donor:recipient DNA ratio can detect rejection in its early stages and before the development of graft dysfunction. AIM 3: Test this approach in lung transplantation and determine whether donor:recipient DNA ratio can differentiate between graft dysfunction due to rejection versus pulmonary infection. If achieved, these specific aims will provide the foundation for validation studies of a donor-specific, genome-based approach to non-invasive, early detection of rejection after solid organ transplantation. PUBLIC HEALTH RELEVANCE: Organ transplantation saves the lives of patients with end-stage organ failure, yet in many cases, the transplanted organ is rejected by the recipient, causing a life-threatening situation. We propose to develop and test a new and innovative approach to the non-invasive, early diagnosis of rejection and graft dysfunction after heart and lung transplantation. By simply monitoring the blood of a transplant recipient for level of the donor genome appearing during follow-up after receiving a transplant, we hope to establish a diagnostic tool that has potential to save hundreds of millions of dollars in health care costs annually, and reduce patient morbidity and mortality.