Novel Technologies to Isolate and Analyze Extrachromosomal DNAs for Diagnostic Applications

摘要/摘要 染色体外环状DNA(EccDNA)元件是物理和拓扑上不同的成分 真核基因组，可促进细胞间拷贝数变异，环境适应，以及 功能基因组多样性。最近，高通量测序已经完成了广泛的基因组图谱 可能的eccDNA序列。尽管取得了这一重大进展，但关于这些细菌的生物发生的信息 环状DNA和这些DNA元件与人类病理之间的因果关系仍然非常 有限的。EccDNA分子的序列、结构和生物学特性构成了一门主要知识 GAP，有关这方面的信息可以导致针对疾病特异性eccDNA结构和功能的策略。 EccDNA代表大范围的分子(~100个到1个MBP)，并与在正常人群中发现的eccDNA共存 体细胞；分离和鉴定不同类别的eccDNA具有它们自己独特的宿主 挑战。为了解决这些知识差距，可以直接询问所有环状DNA物种的方法 这些细胞是非常需要的。 在这份FastTrack SBIR提案中，Phinome，Inc.正在开发一种基于尿液的创新测试方法， 将分离出适合下一代测序和表观遗传学的完整状态的eccDNA分子 特征，并辅以一套生物物理和新的生物信息学方法来识别和 定义这些分子。在第一阶段，Phinology将1)展示Phintics的专有能力 从人类细胞系中分离全面/无偏的eccDNA谱(循环体)的技术；以及2) 展示Phinology的技术强大的测序、映射、组装和信息处理能力 对孤立的循环系统进行分析。在达到第一阶段进行/不进行的里程碑后，在第二阶段中，Phinology将3)进行验证 Phinology公司的技术作为膀胱癌诊断应用的分子平台；4)扩展 将Phinome的技术应用于生物体液样本；以及5)开发一种检测循环的PCR测试 非肌肉浸润性膀胱癌的DNA种类使CLIA认证成为可能。 初步数据已经开始揭示正常体细胞系和组织中的循环系统，胶质母细胞瘤 肿瘤样本和血浆，加强了eccDNA作为固体和液体生物标记物的使用。 活组织检查应用程序。基于eccDNA生物标记物的监测旨在以一种微创策略 确定对治疗的免疫反应，预测复发可能性，并监测残留疾病。

SUMMARY/ABSTRACT Extrachromosomal circular DNA (eccDNA) elements are physically and topologically distinct components of eukaryotic genomes that can contribute to intercellular copy-number variation, environmental adaptations, and functional genomic diversity. Recently, high-throughput sequencing has made extensive genomic mapping of eccDNA sequences possible. Notwithstanding this major advance, information regarding biogenesis of these circular DNAs and causal connections between these DNA elements and human pathologies, remains extremely limited. The sequences, structure, and biological properties of eccDNA molecules constitute a major knowledge gap, information about which can lead to strategies targeting disease-specific eccDNA structure and function. eccDNAs represent a large range of molecules (~100 bp to >1 Mbp) and coexist with eccDNAs found in normal somatic cells; isolating and characterizing different classes of eccDNA comes with their own host of unique challenges. To address these knowledge gaps, methods that can directly interrogate all circular DNA species in the cell are critically needed. In this FastTrack SBIR proposal, Phinomics, Inc. is developing is developing an innovative urine-based test that will isolate eccDNA molecules in an intact state suitable for next-generation sequencing and epigenetic characterization, and is complemented by a suite of biophysical and novel bioinformatic methods to identify and define these molecules. In Phase I, Phinomics will 1) Demonstrate the capability of Phinomics’ proprietary technology to isolate comprehensive/unbiased eccDNA repertoires (circulomes) from human cell lines; and 2) Demonstrate the capability of Phinomics’ technology to robustly sequence, map, assemble, and informatically profile the isolated circulome. Upon meeting Phase I Go/No-Go milestones, in Phase II Phinomics will 3) Validate Phinomics’ technology as a molecular platform for bladder cancer diagnostic applications; 4) Extend the application of Phinomics’ technologies to biological fluid samples; and 5) Develop a PCR test to detect circular DNA species in non-muscle invasive bladder cancer to enable CLIA certification. Preliminary data has begun to illuminate the circulome in normal somatic cell lines and tissues, glioblastoma tumor samples, and plasma, potentiating the use of eccDNA as a valuable biomarker in both solid- and liquid- biopsy applications. Surveillance based on eccDNA biomarkers is aimed at a minimally invasive strategy to determine the immune response to therapies, predict recurrence likelihood, and monitor residual disease.