Super-sensitive detection of universal markers of allograft rejection

同种异体移植排斥通用标记物的超灵敏检测

• 批准号: 9909410
• 负责人: James L Murray
• 金额: $ 24.26万
• 依托单位: GENETAG TECHNOLOGY, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-17 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9909410
• 关键词: Acute; Address; Adrenal Cortex Hormones; Alleles; Allografting; Antibodies; Base Sequence; Binding; Biological Assay; Biological Markers; Biopsy; Blood; Blood specimen; Calcineurin inhibitor; Cancer Patient; Cells; Clinical; Combined Modality Therapy; Companions; DNA; DNA Sequence Alteration; Detection; Development; Discrimination; Doctor of Philosophy; Dose; Early Diagnosis; Epidermal Growth Factor Receptor; FDA approved; Failure; Fluorescence; Frequencies; Gene Frequency; Genes; Gold; Graft Rejection; Grant; Heart Transplantation; Heart-Lung Transplantation; Immunosuppressive Agents; Individual; Kidney; Kidney Transplantation; Label; Life Expectancy; Liver; Lung Transplantation; Malignant neoplasm of lung; Measures; Mediating; Methods; Minor; Molecular Diagnostic Testing; Monitor; Mutation; Non-Small-Cell Lung Carcinoma; Nucleic Acid Amplification Tests; Nucleic Acids; Oncogenic; Operative Surgical Procedures; Organ; Organ Transplantation; Patient Care; Patients; Pharmaceutical Preparations; Phase; Physicians; Plasma; Predictive Value; Preparation; Preventive therapy; Principal Investigator; Probability; Process; Reflex action; Regimen; Research; Risk; Sampling; Sensitivity and Specificity; Single Nucleotide Polymorphism; Site; Small Business Innovation Research Grant; Specimen; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; System; Technology; Temperature; Testing; Time; Tissues; Transplant Recipients; Variant; allograft rejection; base; cell free DNA; cost; diagnosis standard; digital; follow-up; genetic profiling; genetic variant; improved; instrumentation; liquid biopsy; mRNA Expression; medical complication; mutant; next generation sequencing; novel; novel therapeutics; platform-independent; prevent; programs; success; validation studies

Super-sensitive detection of universal markers of allograft rejection Confidential Principal Investigator: Shafer, David A., PhD PROJECT SUMMARY Over 35,000 organ transplants are performed annually in the US, with kidney, liver, heart, and lung transplants being most common. Transplant recipients are normally prescribed immunosuppressants, corticosteroids, or other medications to prevent allograft rejection; however, rejection eventually occurs in up to one third of recipients. Monitoring rejection facilitates timely therapy by switching to a new drug, adding another drug, or adjusting the dose of the current medications. Tissue biopsies are the gold standard for diagnosing allograft rejection, although they are invasive, may result in medical complications, and often yield inadequate specimens. Currently, the AlloMap® test (CareDx) is the only FDA-approved molecular diagnostics test for identifying the risk of acute cell rejection in transplant recipients. The AlloMap® test does not directly measure donor-derived cell-free DNA (ddcfDNA), has a low positive-predictive value, and does not detect antibody- mediated rejection. Thus, a genuine clinical need exists for accurate methods that enable inexpensive, non- invasive “liquid biopsy” biomarker testing via plasma samples to quickly identify subjects at risk of rejection. The overall objective of this project is to develop a real-time PCR SNP profiling test for detecting ddcfDNA in the plasma of transplant recipients, as an early indicator of rejection. Our primary system, the internal DNA- Detection Switch (iDDS) probe system, comprises two interacting components: a fluor-labeled probe, and a quencher-labeled antiprobe that is nearly complementary to the probe. This unique probe system shows superior single-base discrimination over a much wider annealing-temperature range (10–30ºC) than common methods. Recently, we merged our iDDS probe technology with our Wild Terminator (WTx) methods that enable detection of rare mutants (0.1–0.01% frequency) by blocking amplification of the wild-type sequence. The combined method showed greater sensitivity in detecting circulating EGFR variants from lung cancer patients than the FDA-approved cobas® EGFR Mutation Test, v2. Our higher sensitivity and specificity will enable development of platform-independent, liquid biopsy assays. Our Specific Aims are: 1) to develop dual-iDDS probe-based SNP profiling assays for donor-derived SNPs, and 2) to develop WTx-iDDS probe assays for quantitating SNPs in ddcfDNA. In Aim 1, we will develop dual-iDDS probe profiling assays for two SNP variants at each of 20 loci, at sites where the global minor allele frequency is nearly 0.5. Selecting SNPs with nearly equal major/minor allele frequencies will greatly simplify the process of detecting a spike in ddcfDNA levels in the blood of transplant recipients before rejection occurs. In Aim 2, we will develop WTx assays that selectively amplify the 20 targeted SNP alleles, and validate key test features (linearity, analytical sensitivity, comparison with NGS, and precision) required for a 510(k) submission. We will require that each WTx-iDDS probe assay can detect the target SNP at a frequency of 0.1%, which is below the threshold observed in patients before allograft rejection. Success in Phase I will justify expanded Phase II assay-validation studies in preparation for filing for FDA approval.

同种异体拒绝拒绝机密研究员的超敏感检测：Shafer，David A.，PhD 项目摘要 每年在美国进行超过35,000台器官移植，肾脏，肝脏，心脏和肺部进行 移植是最常见的。移植接受者通常是处方的免疫抑制剂， 皮质类固醇或其他药物以防止同种异体移植排斥；但是，拒绝最终发生在 三分之一的接收者。通过改用新药，及时添加另一种 药物或调整当前药物的剂量。组织活检是诊断的黄金标准 同种异体拒绝虽然具有侵入性，但可能导致医疗并发症，并且常常导致不足 标本。目前，Allomap®测试（Caredx）是唯一的FDA批准分子诊断测试 确定移植受者急性细胞排斥的风险。 Allomap®测试未直接测量 供体衍生的无细胞DNA（DDCFDNA）的阳性预测值低，并且不检测到抗体 - 介导的拒绝。这是对准确方法的真正临床需求 通过血浆样品进行侵入性的“液体活检”生物标志物测试，以快速识别有排斥风险的受试者。 该项目的总体目的是开发用于检测DDCFDNA的实时PCR SNP分析测试 在移植受者的血浆中，作为排斥反应的早期指标。我们的主要系统，内部DNA- 检测开关（IDD）探针系统包括两个相互作用的组件：一个荧光标记的探针和一个 Quencher标记的抗果杆几乎是探针的完整性。这个独特的探针系统显示出优越的 比常见方法比较宽的退火温度范围（10–30ºC）的单基歧视。 最近，我们将IDDS探测技术与野生终结器（WTX）方法合并，以实现检测 通过阻断野生型序列的扩增，稀有突变体（0.1-0.01％频率）组合 方法表明，在检测肺癌患者的循环EGFR变体方面比 FDA批准的Cobas®EGFR突变测试，V2。我们更高的敏感性和特异性将使发展 与平台无关的，液体活检测定法。我们的具体目的是：1）开发基于双IDDS探测的SNP 供体衍生的SNP的分析测定和2）开发WTX-IDDS探测测定法，以定量SNP DDCFDNA。在AIM 1中，我们将针对20个基因座中的每个SNP变体开发双IDDS探针分析测定法 全球次要等位基因频率几乎为0.5的站点。选择几乎相等/小等位基因的SNP 频率将大大简化检测移植血液中DDCFDNA水平的尖峰的过程 发生拒绝之前的接收者。在AIM 2中，我们将开发WTX测定法，以选择性扩大20个目标 SNP等位基因和验证关键测试特征（线性，分析灵敏度，与NGS的比较以及精度） 提交510（k）所需。我们将要求每个WTX-IDDS探针测定可以检测到目标SNP 在同种异体移植排斥前观察到的0.1％的频率低于患者的阈值。成功 第一阶段将证明扩大的II期测定验证研究是合理的，以准备申请FDA批准。