Rapid, simple, and ultrasensitive quantitation of KRAS ctDNA at the point of care using CRISPR/Cas amplification and digital resolution biosensor microscopy

使用 CRISPR/Cas 扩增和数字分辨率生物传感器显微镜在护理点快速、简单且超灵敏地定量 KRAS ctDNA

• 批准号: 10709211
• 负责人: Brian T. Cunningham
• 金额: $ 39.15万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10709211
• 关键词: Address; Aftercare; Base Sequence; Biological; Biological Assay; Biological Markers; Biosensing Techniques; Biosensor; Blood; Blood Plasma Volume; Calibration; Cancer Detection; Cell physiology; Clinical; Clonal Evolution; Clustered Regularly Interspaced Short Palindromic Repeats; Colorectal Cancer; Complement; DNA; DNA Sequence; DNA Sequence Alteration; Detection; Development; Diagnosis; Diagnostic; Diagnostic tests; Discrimination; Disease; Disease remission; Effectiveness; Elements; FDA approved; Fluorescence; Gene Frequency; Genes; Genomics; Guide RNA; Hour; Human; KRAS2 gene; Laboratories; Link; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of pancreas; Measurement; Measures; Methods; Microscopy; Molecular; Molecular Abnormality; Monitor; Mutation; Mutation Detection; Nucleic Acid Probes; Nucleic Acids; Office Visits; Performance; Pharmaceutical Preparations; Plasma; Process; Protocols documentation; RNA Probes; Rapid diagnostics; Recommendation; Recurrence; Reference Standards; Residual Neoplasm; Resolution; Sampling; Screening for cancer; Selection for Treatments; Single-Stranded DNA; Slide; Specificity; Specimen; Surface; System; Technology; Testing; Time; Tissues; Tube; Tumor Biology; Validation; Variant; assay development; cancer biomarkers; clinical efficacy; clinical practice; clinically relevant; comparative; cost; design; design,build,test; detection limit; detection sensitivity; digital; follow-up; genetic variant; genomic biomarker; improved; innovation; insight; instrument; liquid biopsy; multiplex detection; mutant; nanoGold; next generation sequencing; novel; nucleic acid detection; photonics; point of care; point-of-care detection; point-of-care diagnostics; portability; pressure; screening; sensor technology; targeted treatment; technology platform; tool; treatment effect; tumor; tumor DNA

Abstract While a growing arsenal of drugs is available to treat specific molecular abnormalities across cancers, therapy effectiveness can now be predicted by detecting specific genomic circulating tumor DNA (ctDNA) in plasma. While next-generation sequencing (NGS) can provide a comprehensive readout of genomic tumor variants that may provide biological and clinical efficacy insights, its cost, complexity, and sample-to-answer timeframe are not compatible with frequent, routine, point of care diagnostics. Meanwhile, currently available laboratory-based methods for quantifying strategically-selected ctDNA biomarkers in plasma for liquid biopsy lack sensitivity, multiplexing, and workflow simplicity required for clinical needs. A genomic liquid biopsy that can be rapidly performed in a clinical setting in the timeframe of an office visit offers a compelling alternative for identifying the presence, absence, and concentration changes in circulating nucleic acid molecules whose specific base sequences represent mutations that drive cancer-associated cellular processes. Such an approach would enable therapy selection to be performed at the earliest time while facilitating more frequent remission monitoring. To address the gaps in current technology, we seek to develop and rigorously validate a novel assay method called “Activate, Cleave, Capture, and Count” (AC3) that combines two innovative elements. First, we apply a recently-demonstrated photonic crystal (PC) biosensor microscopy technology with digital resolution capability for quantifying surface-captured gold nanoparticle (AuNP) tags. Second, we utilize the CRISPR/Cas system with target-specific guide RNA probes that selectively activate cleavage of ssDNA tethers linking AuNPs to a surface, generating many released AuNPs for each ctDNA molecule. The released AuNPs are subsequently captured on a PC biosensor, where they are digitally counted. Our ”amplify-then-digitize” strategy offers a compelling alternative to digital PCR-based technologies while also circumventing the limitations inherent with thermal amplification, microdroplet partitioning, and fluorescence-based detection. Based upon preliminary results for the detection of cancer-associated ctDNA, AC3 offers a detection limit of 50 zM and a measurement of mutant allele frequency of <0.001%. Importantly, AC3 utilizes a small and inexpensive (~ $7K) detection instrument. In this project, we will apply AC3 for characterization of plasma ctDNA biomarkers across six mutations and characterize performance using spiked-in calibration standards, and in banked human plasma samples. We will rigorously characterize the sensitivity, selectivity, and repeatability of AC3 compared to droplet digital PCR (ddPCR). We envision AC3 as a complement to tissue-based NGS, applied to routine initial cancer screening for therapy selection, monitoring the effects of treatments, and as a remission monitoring tool. Compared with alternatives, the inherently greater sensitivity of AC3 offers opportunities to perform earlier cancer detection, integrate higher levels of multiplexing, and reduce plasma volume requirements.

摘要 虽然越来越多的药物可用于治疗癌症中的特定分子异常，但治疗 现在可以通过检测血浆中特定的基因组循环肿瘤DNA（ctDNA）来预测有效性。 虽然下一代测序（NGS）可以提供基因组肿瘤变体的全面读数， 可以提供生物学和临床疗效的见解，其成本，复杂性和样本到答案的时间范围是 与频繁、常规、护理点诊断不兼容。与此同时，目前可用的基于实验室的 用于定量血浆中策略性选择的ctDNA生物标志物以进行液体活组织检查的方法缺乏灵敏度， 多路复用和临床需求所需的工作流程简单性。一种基因组液体活检， 在诊所就诊的时间范围内在临床环境中进行，为识别 循环核酸分子的存在、不存在和浓度变化，所述循环核酸分子的特异性碱基 序列代表驱动癌症相关细胞过程的突变。这种做法将 使治疗选择能够在最早的时间进行，同时促进更频繁的缓解 监测.为了解决当前技术的差距，我们寻求开发并严格验证一种新的检测方法， 一种称为“激活、切割、捕获和计数”（AC 3）的方法，它结合了两个创新元素。一是 应用最近演示的具有数字分辨率的光子晶体（PC）生物传感器显微镜技术 用于定量表面捕获的金纳米颗粒（AuNP）标签的能力。其次，我们利用CRISPR/Cas 具有选择性激活连接AuNP的ssDNA系链的切割的靶特异性引导RNA探针的系统 到表面，为每个ctDNA分子产生许多释放的AuNP。释放的AuNP随后被 在PC生物传感器上捕获，在那里它们被数字计数。我们的“先放大后放大”战略提供了 数字PCR技术的替代方案，同时也规避了 热扩增、微滴分配和基于荧光的检测。根据初步 对于癌症相关ctDNA的检测结果，AC 3提供50 zM的检测限和 突变等位基因频率<0.001%。重要的是，AC 3采用了一种小型且廉价（约7000美元）的检测方法， 仪器在这个项目中，我们将应用AC 3来表征六种血浆ctDNA生物标志物， 突变，并使用加标校准标准品和库存人血浆表征性能 样品我们将严格表征AC 3与液滴相比的灵敏度、选择性和可重复性 数字PCR（ddPCR）。我们设想AC 3作为基于组织的NGS的补充，应用于常规的初始癌症 筛选治疗选择，监测治疗效果，并作为缓解监测工具。 与替代品相比，AC 3固有的更高灵敏度为早期癌症提供了机会 检测，集成更高水平的多路复用，并降低血浆体积要求。