miRNA HD Array Platform

描述(申请人提供)：本申请是第一阶段申请的第一次重新提交。NIAID对SBIR/STTR应用的一个特别感兴趣的领域是“对生物恐怖主义病原体的宿主免疫防御的生物标记物”。利用在白细胞中表达的miRNA(MiRNA)作为生物标志物来筛选感染是新颖的，并且潜在地非常有效，因为不仅宿主细胞miRNA在感染时发生变化，而且RNA和DNA病毒都调节在宿主细胞中表达的miRNA。更新颖的是一种同时测量miRNA和mRNAs生物标记物的方法。这一阶段的应用将开发和验证一种仅裂解、定量和高重复性的高密度阵列miRNA平台(HD qNPA miRNA分析)，该平台基于对mRNA基因表达的核酸酶保护分析，即HTG销售的定量核酸酶保护分析(qNPA“)，用于测量miRNA。自第一次提交以来，HTG已将qNPA mRNA分析修改为高密度阵列平台，并正在验证基于qNPA的定制微板miRNA分析。该检测方法将适用于高密度平台，并在第一阶段计划中得到验证。QNPA只需要样品裂解(不提取mRNA)，这使这成为一种新的miRNA定量方法。目前用于测量miRNA的方法存在问题，部分原因是由于提取效率的不确定性，现有方法难以从固定组织中测量mRNA(更少的miRNA)，以及当前高密度阵列和miRNA测量方法的较差的定量性能。HD qNPA miRNA分析的性能将首先在细胞、固定组织、全血、支气管灌洗、痰和气管抽吸物样本中得到验证。预计从这些复杂的临床样本中，整个检测CV将为&lt；20%(理想情况下为&lt；10%)，能够识别&lt；1.4倍(理想情况下为1.2倍)的变化，并且这种灵敏度将使&lt；60毫升全血或最少5,000个细胞的使用成为可能。将测试一组来自被诊断为脓毒症的患者的血液样本，以及一组来自弥漫性大B细胞淋巴瘤(DLBCL)患者的存档福尔马林固定石蜡包埋活检组织，这些患者随后接受了化疗加抗CD20单抗Ritixan。这两项临床研究将验证该平台的miRNA发现概念，并证明可以从这些样本中识别出miRNA的定量差异图谱(与正常或对照样本相比)，提供多组假定的miRNA生物标志物，作为治疗反应(对于DLBCL)或启动“早期目标导向治疗”(对于脓毒症)的预测筛查。在第二阶段，将对DLBCL和脓毒症的样本测试集进行评估，并将扩展该方法，以识别和验证对A、B和C类药物的感染筛查有用的白细胞miRNA签名，解决是否可以识别生物特有的签名，或者miRNA签名是针对病原体类别，还是仅针对感染的通用签名。鉴定出的miRNA还将作为抗感染药物的靶标进行评估。与公共卫生相关：这项涉及HTG和亚利桑那大学的为期2年的第一阶段STTR申请正在(首次)重新提交，以解决NIAID特别感兴趣的一个领域，即“对生物恐怖主义病原体的宿主免疫防御的生物标记物”。我们建议开发和验证一种仅裂解、定量和高重复性的高密度阵列microRNA(MiRNA)平台-HD qNPA miRNA分析-基于对mRNA基因表达的核酸酶保护分析，即HTG推出的基于微孔板的定量核酸酶保护分析(qNPA“)。我们建议证明这个新的HD qNPA miRNA平台可以从临床样本(血液、支气管灌洗、痰、气管抽吸物、福尔马林固定的石蜡包埋组织)的宿主细胞中识别miRNA组，并特异性地验证其作为脓毒症和弥漫性大B细胞淋巴瘤的筛查方法的性能。使用miRNA作为传染病的生物标志物是新颖的，而且可能非常有效，因为不仅宿主细胞的miRNA在感染后发生变化，而且RNA和DNA病毒都调节在宿主细胞中表达的miRNA。此外，这一应用的其他重要和新颖的方面是，该分析将同时测量miRNA和mRNA签名，它将不需要提取(对于miRNA来说是有问题的)，并且它应该在灵敏度、精密度、重复性和从临床相关样本(如全血和固定组织)中测量miRNA的能力方面提供卓越的性能。在第二阶段，该平台将用于识别宿主细胞miRNA特征，这些特征有助于筛选A、B和C类病原体的感染，并可用作药物发现的目标。

DESCRIPTION (provided by applicant): This application is a first resubmission of a Phase I application. An area of special interest to NIAID for SBIR/STTR applications is "Biomarkers of Host Immune Defense to Bioterrorism Pathogens". The use of microRNA's (miRNA) expressed in white blood cells as biomarkers to screen for infection is novel and potentially very powerful because not only do host cell miRNA's change upon infection, but both RNA and DNA viruses regulate miRNA that are expressed in the host cell. Even more novel is an assay that measures both miRNA and mRNA biomarkers simultaneously. This Phase I application will develop and validate a lysis-only, quantitative and highly reproducible high density array miRNA platform (the HD qNPA miRNA assay) based on adapting a nuclease protection assay of mRNA gene expression, the quantitative Nuclease Protection Assay (qNPA") marketed by HTG, to measure miRNA. Since the first submission, HTG has adapted the qNPA mRNA assay to a high density array platform and is in the process of validating a qNPA-base custom microplate miRNA assay. This assay will be adapted to the high density platform and validated in this Phase I program. qNPA only requires sample lysis (no extraction of the mRNA), making this a novel approach to miRNA quantification. Current methods for measuring miRNA are problematic, in part because of uncertainties regarding the efficiency of extraction, the difficulty current methods have measuring mRNA (much less miRNA) from fixed tissue, and the poor quantitative performance of current high density arrays and miRNA measurement methods. Performance of the HD qNPA miRNA assay will be first validated for samples of cells, fixed tissues, whole blood, bronchial lavage, sputum, and tracheal aspirates. The expectation is that whole assay CV's will be <20% (ideally <10%) from these complex clinical samples, enabling the identification of changes of <1.4-fold (ideally <1.2-fold), and that sensitivity will enable the use of <60 ml whole blood or as few as 5,000 cells. A training set of blood samples from patients diagnosed with sepsis and a training set of archived formalin fixed paraffin embedded biopsies from patients with diffuse large B cell lymphoma (DLBCL), who subsequently received chemotherapy plus anti-CD20 monoclonal Ritixan, will be tested. These two clinical studies will validate the miRNA discovery concept of the platform and demonstrate that quantitative differential profiles of miRNA can be identified from such samples (compared to normal or control samples), providing sets of putative miRNA biomarkers useful as a predictive screen for response to therapy (in the case of DLBCL) or for initiating "early goal-directed therapy" (in the case of sepsis). In Phase II, test sets of samples for DLBCL and sepsis will be evaluated, and the method will be extended to identify and validate white cell miRNA signatures useful for screening for infection by Category A, B, and C agents, addressing whether organism-specific signatures can be identified, or whether the miRNA signatures will be specific for the class of pathogen, or only generic for infection. The identified miRNA will also be evaluated for utility as targets for anti-infectives. PUBLIC HEALTH RELEVANCE: This 2-year Phase I STTR application involving HTG and the University of Arizona is being resubmitted (for the first time) to address an area of special interest to NIAID for "Biomarkers of Host Immune Defense to Bioterrorism Pathogens". We propose to develop and validate a lysis-only, quantitative and highly reproducible high density array microRNA (miRNA) platform - the HD qNPA miRNA assay - based on adapting a nuclease protection assay of mRNA gene expression, the microplate-based quantitative Nuclease Protection Assay (qNPA") marketed by HTG. We propose to demonstrate that this novel HD qNPA miRNA platform can identify sets of miRNA from host cells within clinical samples (blood, bronchial lavage, sputum, tracheal aspirate, formalin fixed paraffin embedded tissue), and to specifically validate performance as a screening method for sepsis and diffuse large B cell lymphoma. The use of miRNA as biomarkers of infectious diseases is novel and potentially very powerful since not only do host cell miRNA's change upon infection, but both RNA and DNA viruses regulate miRNA that are expressed in the host cell. Additionally, other significant and novel aspects of this application are that the assay will measure both miRNA and mRNA signatures, that it will not require extraction (problematic for miRNA), and that it should provide superior performance in terms of sensitivity, precision, reproducibility and ability to measure miRNA from clinically relevant samples (such as whole blood and fixed tissue). In Phase II, this platform will be applied to identify host cell miRNA signatures useful for screening for infection by Category A, B and C pathogens, and useful as targets for drug discovery.