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

描述(由申请人提供)：这个拟议的第一阶段SBIR项目将把学术界开发的定向测序技术RASL-Seq转移给255Xpress，Inc.，并证明其商业化作为药物发现和高通量筛选(HTS)平台的可行性。这项技术使用寡聚连接、扩增和下一代测序进行定量，一次检测细胞裂解产物中的数百个RNA靶标。这种高度多元化的HTS检测方法测量一组基因和途径，而不是单一目标，并将结果表示为单一分数。因此，可以在不监控可用药靶点的情况下检测药物反应，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非靶标的不良反应，而不是在临床开发或上市后的后期发现，在那里唯一的选择是停止开发或停用药物。鉴于三分之一的晚期失败是由于本可以避免的意想不到的非靶向效应所致，RASL-SEQ将在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。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、硬化试剂配方和分析流程，以及内部和外部分析控制。我们将建立性能衡量标准(重复性、动态范围、错配率和控制行为)，并进行稳健性和验证性研究。我们还将演示HTS和QSAR优化分析的可行性，该分析测量10到1,000个基因的表达，重复性CV为15%。RASL-Seq可以完全自动化，并允许在测序前汇集样品，我们将展示这将把HTS的每个样品的总成本降低到每个样品不到3美元，这一价格点对于多路分析的商业采用至关重要。RASL-Seq还可用于重新调整药物用途，或通过分析识别签名，然后重新测试类似物和重新筛选，在临床开发中因非目标效应而导致先导失败的项目。迫切需要药物作用机制的新靶点，而这个技术平台提供了解锁以前无法用药的作用机制的能力，从而理性地发现药物。

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.