METHYL-SENTRY: Proposed feasibility study of a nanopore diagnostic tool with rapid automated measurement of cell free DNA methylation state for clinical cancer evaluation

METHYL-SENTRY：拟议的纳米孔诊断工具的可行性研究，可快速自动测量无细胞 DNA 甲基化状态，用于临床癌症评估

• 批准号: 10708833
• 负责人: David John Niedzwiecki
• 金额: $ 16.33万
• 依托单位: GOEPPERT, LLC
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-22 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10708833
• 关键词: Apoptosis; Base Composition; Binding; Binding Proteins; Biological Assay; Biological Markers; Blood; Cancer Detection; Cancer Patient; Cell Fraction; Clinical; Cytosine; DNA; DNA Methylation; DNA Sequence; Detection; Diagnostic; Dinucleoside Phosphates; Discrimination; Disease Progression; Early Diagnosis; Engineering; Evaluation; Feasibility Studies; Frequencies; Gene Expression; Genome; Goals; Green Fluorescent Proteins; Guanine; Human Genome; Hypermethylation; Island; Label; Length; Malignant Neoplasms; Measurement; Measures; Methods; Methylation; Morbidity - disease rate; Mutation; Necrosis; Noise; Normal Cell; Nucleotides; Outcome; Patients; Pattern; Pore Proteins; Preparation; Promoter Regions; Protein Binding Domain; Proteins; Research Personnel; Sampling; Screening for cancer; Series; Serum; Signal Transduction; Site; Specificity; System; Technology; Testing; Urine; Work; cancer biomarkers; cancer therapy; cancer type; cell free DNA; clinical efficacy; commercialization; coomassie Brilliant Blue; detection method; detection platform; diagnostic tool; improved; improved outcome; liquid biopsy; nanopore; neoplastic cell; promoter; screening; single molecule; solid state; tumor; tumor DNA; tumor progression

METHYL-SENTRY: Proposed feasibility study of a nanopore diagnostic tool to detect hypermethylated biomarkers in cell free DNA indicative of cancer state Early detection of cancer is correlated with improved outcomes and reduced morbidity due to more timely treatment. Ideally, detection strategies should be non-invasive, rapid, and easy to deploy in a clinical setting. Circulating cell-free DNA (cfDNA) is released by normal cells, however, in cancer patients a portion of cfDNA comes from tumor cells (circulating-tumor DNA or ctDNA). As tumor cells divide faster than normal cells, cancer patients typically have a high level of cfDNA in serum because of necrosis or apoptosis, with a greater proportion of cfDNA attributable to tumors. Detection of the ctDNA fraction of cfDNA presents an excellent opportunity for a non-specific, blood- or urine-based screening, referred to as a “liquid biopsy”. One of the most readily detected changes in the DNA of cancer patients is alterations in the pattern of genome methylation, a significant control mechanism of gene expression. However, there is limited diagnostic information from detecting an increase in cfDNA, and later approaches targeted ctDNA and cancer-specific alterations to this DNA. Here we propose the use of solid-state nanopores for detection of hypermethylated cytosine adjacent guanine (CpG) dinucleotide clustered islands (CGI) in cfDNA as a sensitive and non-specific technique for detection of cancer from blood or urine samples. As with protein nanopores, solid-state nanopores allow single molecule measurements with translocation of the DNA strand through the pore resulting in a unique electrical signal. Sequences will be evaluated in the presence of methyl binding domain (MBD) protein for selectively binding to methylated CpG domains and further amplification of nanopore derived quantification signal. In our proposed method, the electrical signature is representative of the entire molecule, instead of a single nucleotide, for detection of hypermethylated CGIs as a cancer biomarker. The proposed work is supported by three Technical Objectives (TOs). TO1 is to determine the sensitivity of the method for the number of methylated CpG per CGI fragment. TO2 will determine the ability of our method to detect an increase in hypermethylation of CGIs above the background of partially methylated cfDNA in a control sample. TO3 will determine the feasibility and sensitivity of this system in a series of clinical cancer mimic samples. In contrast to previously developed conventional methods, our proposed method would be fast, sensitive, and independent of the underlying DNA sequence so that it could be used for cancer detection regardless of mutations in the target sequence. This solid-state based method can ultimately be commercialized into a simple to use clinical setting general cancer detection and treatment progression diagnostic.

METHYL-SENTRY：拟议的纳米孔诊断工具的可行性研究，以检测 无细胞DNA中高甲基化生物标志物指示癌症状态 癌症的早期发现与改善的结果和降低的发病率相关，因为更及时的治疗可以减少癌症的发生。 治疗理想情况下，检测策略应该是非侵入性的，快速的，并且易于在临床环境中部署。 循环无细胞DNA（cfDNA）由正常细胞释放，然而，在癌症患者中， cfDNA来自肿瘤细胞（循环肿瘤DNA或ctDNA）。由于肿瘤细胞分裂的速度比 正常细胞，癌症患者通常由于坏死或凋亡而在血清中具有高水平的cfDNA， 其中较大比例的cfDNA可归因于肿瘤。cfDNA的ctDNA部分的检测 为非特异性的、基于血液或尿液的筛查提供了极好的机会，称为“液体 活组织检查”。癌症患者DNA中最容易检测到的变化之一是模式的改变， 基因组甲基化是基因表达的重要控制机制。然而， 通过检测cfDNA的增加获得诊断信息，以及后来的方法靶向ctDNA， 癌症特异性的DNA改变 在这里，我们提出了使用固态纳米孔检测超甲基化胞嘧啶相邻 cfDNA中的鸟嘌呤（CpG）二核苷酸簇集岛（CGI）作为一种敏感和非特异性的技术， 从血液或尿液样本中检测癌症。与蛋白质纳米孔一样，固态纳米孔允许 单分子测量与易位的DNA链通过孔导致在一个独特的 电信号。将在甲基结合结构域（MBD）蛋白存在下评价序列， 选择性结合甲基化的CpG结构域，并进一步扩增纳米孔衍生的 量化信号在我们提出的方法中，电子签名代表了整个 在一些实施方案中，使用一种分子而不是单个核苷酸来检测作为癌症生物标志物的高甲基化CGI。 这项工作得到了三个技术目标（TO）的支持。TO 1用于确定灵敏度 每个CGI片段的甲基化CpG数量的方法。TO 2将决定我们 检测CGI超甲基化增加超过部分甲基化背景的方法 对照样品中的cfDNA。TO 3将确定该系统在一系列测试中的可行性和灵敏度， 临床癌症模拟样品。 与以前开发的常规方法相比，我们提出的方法将是快速，灵敏， 并且不依赖于潜在的DNA序列，因此它可以用于癌症检测， 目标序列中的突变。这种基于固态的方法最终可以商业化为一种新的方法。 使用简单的临床设置一般癌症检测和治疗进展诊断。