nCounter Prep Station and the Digital Analyzer

nCounter Prep Station 和数字分析仪

• 批准号: 7793214
• 负责人: Laurence J.N. Cooper
• 金额: $ 23.58万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-26 至 2011-01-25
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7793214
• 关键词: Active immunity; Animals; Applications Grants; Biological Assay; Biological Response Modifier Therapy; Cancer Immunology Science; Cells; Clinical; Detection; Enzymes; Flow Cytometry; Formalin; Gene Expression; Gene Expression Profile; Genes; Homing; Human; Image; Immune; Immunophenotyping; Immunotherapeutic agent; Immunotherapy; Infusion procedures; Institution; Malignant Neoplasms; Measurement; Messenger RNA; Modern Medicine; Molecular; Molecular Profiling; Paraffin Embedding; Patients; Phenotype; RNA; Reaction; Research; Research Personnel; Role; Sampling; Serological; Specificity; System; Systems Analysis; Technology; Testing; Therapeutic; Tissue Sample; Tissues; Transcript; University of Texas M D Anderson Cancer Center; Whole Blood; Work; base; conventional therapy; design; digital; improved; mRNA Expression; new technology; next generation; novel; programs; protein expression; single molecule; tissue/cell culture; tumor

DESCRIPTION (provided by applicant): Cell-based immunotherapies hold great promise for treatment of malignancies, yet they are among the greatest challenges to develop and implement in modern medicine. There are typically no or few markers such as distinct clinical features, specific morphological changes by imaging, or serological identifiers, which can predict efficacy or even therapeutic potential of these biologic therapies. Despite these hurdles, and the challenge of generating cell-based immunotherapies from within not-for-profit academic institutions, immune- based therapies are being developed and tested in humans based on the premise and promise of their specificity and ability to eradicate tumor using effector mechanisms that are non-overlapping with conventional therapies. The Center for Cancer Immunology Research (CCIR) at University of Texas (UT) M.D. Anderson Cancer Center (MDACC) has an active program in designing and implementing clinical-grade immunotherapies based on enhancing passive and active immunity. Currently, the phenotype of cells infused and cells recovered from patients (and animals) are characterized by flow cytometry for protein expression and a limited analysis of expressed genes. However, it is vital to be able to classify immune genes that are differentially expressed before and after immunotherapy to gauge the therapeutic potential of the treatment and to undertake the rational design of next generation immunotherapeutics with predicted improved biologic effect. The two current approaches to profiling immune gene expression, microanalysis and Q-PCR, have a limited role in enhancing the potential of immunotherapy due to the large amount of material needed, with the resultant bias that occurs when mRNA is limiting and does not cover the array's dynamic concentration range, or the bias occurring with enzymatic amplification. Simply put, comprehensive mRNA expression profiling to assess activation status, homing potential, and immunophenotype, is not possible on the immune cells recovered after infusion due to the small amount of recovered tissue. Fortunately, new technology is now available using the nCounter Analysis System from NanoString Technologies, Inc. (Seattle, WA). This system uses no enzymes or amplification steps, yet can analyze hundreds of genes in a single assay with high precision, even at low expression levels using samples directly obtained from cell culture, tissue, or whole blood lysates without sample purification and significantly can assay total purified RNA derived from formalin- fixed paraffin-embedded (FFPE) tissue samples. This system employs a novel digital technology to enable direct multiplexed measurement of gene expression while offering a high level of sensitivity and precision, including detection of fractional fold-change differences. The technology uses molecular barcodes and single molecule imaging to detect and count hundreds of unique transcripts in a single reaction. The fully automated system is easy to use and is ideally suited for researchers \on this grant application who wish to validate gene expression signatures, working with small amounts of starting material and studying defined gene sets.

描述（由申请人提供）： 基于细胞的免疫疗法在治疗恶性肿瘤方面具有巨大的前景，但它们是现代医学开发和实施的最大挑战之一。通常没有或很少有标志物，例如独特的临床特征、通过成像的特定形态学变化或血清学标识符，其可以预测这些生物疗法的功效或甚至治疗潜力。尽管存在这些障碍，以及在非营利学术机构内产生基于细胞的免疫疗法的挑战，基于免疫疗法的特异性和使用与常规疗法不重叠的效应机制根除肿瘤的能力的前提和前景，正在人类中开发和测试基于免疫的疗法。德克萨斯大学癌症免疫学研究中心（CCIR）安德森癌症中心（MDACC）有一个积极的计划，设计和实施基于增强被动和主动免疫的临床级免疫疗法。目前，输注的细胞和从患者（和动物）回收的细胞的表型通过用于蛋白质表达的流式细胞术和表达基因的有限分析来表征。然而，能够对免疫治疗前后差异表达的免疫基因进行分类，以评估治疗的治疗潜力，并合理设计具有预期改善的生物学效应的下一代免疫治疗药物，这一点至关重要。目前的两种分析免疫基因表达的方法，微量分析和Q-PCR，由于需要大量的材料，在增强免疫治疗的潜力方面作用有限，当mRNA是有限的并且不覆盖阵列的动态浓度范围时会产生偏差，或者酶促扩增会产生偏差。简单地说，由于回收的组织量小，因此在输注后回收的免疫细胞上不可能进行全面的mRNA表达谱分析以评估活化状态、归巢潜力和免疫表型。幸运的是，现在可以使用NanoString Technologies，Inc.的nCounter分析系统获得新技术。（华盛顿州西雅图）。该系统不使用酶或扩增步骤，但可以在单次测定中以高精度分析数百个基因，甚至在低表达水平下使用直接从细胞培养物、组织或全血裂解物获得的样品而无需样品纯化，并且显著地可以测定源自福尔马林固定石蜡包埋（FFPE）组织样品的总纯化RNA。该系统采用了一种新的数字技术，能够直接多重测量基因表达，同时提供高水平的灵敏度和精度，包括检测分数倍数变化差异。该技术使用分子条形码和单分子成像在单个反应中检测和计数数百个独特的转录本。全自动系统易于使用，非常适合希望验证基因表达特征的研究人员，他们希望使用少量起始材料并研究定义的基因集。