Cell Lysates to Sequencing Reads: A Multiplex Gene Expression Profiling Tool for

细胞裂解物到测序读取：多重基因表达分析工具

• 批准号: 8522760
• 负责人: Joanne Mulligan Yeakley
• 金额: $ 33.74万
• 依托单位: BIOSPYDER TECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-23 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8522760
• 关键词: Academia; Acute; Address; Adoption; Adverse effects; Algorithms; Androgens; Area; Automation; Behavior Control; Biological Assay; Biological Models; Blinded; Buffers; Businesses; Cells; Chemicals; Chemistry; Clinical; Computer software; Cytolysis; Data; Data Analyses; Development; Dose; Drug Formulations; Drug Industry; Early identification; Enzymes; Failure; Gene Expression Profiling; Genes; LNCaP; Laboratories; Ligation; Marketing; Measurable; Measurement; Measures; Methods; Molecular Profiling; Monitor; Oligonucleotides; Pathway interactions; Performance; Pharmaceutical Preparations; Phase; Price; Process; Protocols documentation; Quality Control; Quantitative Structure-Activity Relationship; RNA; RNA annealing; Reading; Reagent; Reproducibility; Sampling; Sensitivity and Specificity; Series; Shipping; Ships; Site; Small Business Innovation Research Grant; Specificity; Staging; Technology; Testing; Therapeutic; Time; Titrations; Transfer RNA; analog; base; combinatorial; commercialization; cost; design; drug development; drug discovery; drug efficacy; drug mechanism; flexibility; high throughput screening; improved; innovation; meetings; next generation sequencing; novel; novel strategies; performance tests; post-market; programs; public health relevance; response; screening; single molecule; success; tool; validation studies

DESCRIPTION (provided by applicant): This proposed Phase I SBIR project will transfer RASL-Seq, a targeted sequencing technology developed in academia, to a small business, 255Xpress, Inc., and demonstrate feasibility for its commercialization as a platform for drug discovery and high throughput screening (HTS). The technology detects hundreds of RNA targets at once in cell lysates, using oligo ligation, amplification, and next-generation sequencing for quantitation. This highly multiplexed HTS assay measures sets of genes and pathways, rather than single targets, and expresses the result as a single score. As a result, drug responses can be detected without monitoring the druggable target, and the technology can be used to assess efficacy and side effects at the same time. This multiplexed profiling approach will enable the identification of off-target adverse effects early in the drug discovery process, rather than being discovered late in clinical development or post marketing where the only option is to stop development or withdraw the drug. Given that 1/3rd of late stage failures are due to unanticipated off- target effects that could have been avoided, RASL-Seq will have a significant impact across all therapeutic areas, increasing the efficiency, cost, and success of getting drugs to market. To demonstrate commercial feasibility, we intend to develop robust probe design and data analysis methods, hardened reagent formulations and assay processes, and internal and external assay controls. We will establish performance measures (reproducibility, dynamic range, mis-ligation rates, and the behavior of controls), and conduct robustness and verification studies. We will also demonstrate the feasibility of HTS and QSAR optimization assays measuring the expression of 10 to 1,000 genes with reproducibility CV's of <15%. RASL-Seq can be fully automated and permits the pooling of samples before sequencing, which we will demonstrate will reduce total cost per sample for HTS to less than $3 per sample, a price point that is critical for commercial adoption of multiplexed assays. RASL-Seq can also be used to repurpose drugs, or to salvage programs where the leads fail in clinical development due to off-target effects, by profiling to identify a signature and then re-testing analogs and re-screening. There is an acute need for new targets for drug mechanisms of action, and this technology platform provides the capability to unlock previously undruggable mechanisms of action to rational drug discovery.

描述（由申请人提供）：该拟议的I期SBIR项目将把RASL-Seq（一种在学术界开发的靶向测序技术）转移给一家小型企业255 Xpress，Inc.，并证明其作为药物发现和高通量筛选（HTS）平台商业化的可行性。该技术使用寡核苷酸连接、扩增和下一代测序进行定量，同时检测细胞裂解物中的数百种RNA靶标。这种高度多重的HTS测定法测量多组基因和途径，而不是单个靶标，并将结果表示为单个评分。因此，药物反应可以在不监测可药用靶标的情况下检测到，并且该技术可以用于同时评估疗效和副作用。这种多重分析方法将能够在药物发现过程的早期识别脱靶不良反应，而不是在临床开发或上市后发现，此时唯一的选择是停止开发或撤回药物。鉴于1/3的晚期失败是由于本可以避免的意外脱靶效应，RASL-Seq将对所有治疗领域产生重大影响，提高药物上市的效率、成本和成功率。为了证明商业可行性，我们打算开发稳健的探针设计和数据分析方法，硬化试剂配方和测定过程，以及内部和外部测定对照。我们将建立性能指标（再现性、动态范围、错误连接率和对照行为），并进行稳健性和验证研究。我们还将证明HTS和QSAR优化测定法测量10至1，000个基因表达的可行性，重现性CV <15%。RASL-Seq可以完全自动化，并允许在测序前合并样品，我们将证明这将使HTS的每个样品的总成本降低到每个样品不到3美元，这一价格点对于多重测定的商业采用至关重要。RASL-Seq还可用于重新利用药物，或挽救因脱靶效应导致临床开发失败的项目，方法是通过分析以识别特征，然后重新测试类似物并重新筛选。迫切需要药物作用机制的新靶标，并且该技术平台提供了解锁先前不可抵抗的作用机制以合理药物发现的能力。