Gene silencing by optoinjection and RNA Interference

描述（由申请人提供）：RNA干扰（RNAi）是一种使用dsRNA模板的细胞内酶复合物介导的特异性基因沉默方法，由于其比旧的反义方法具有重要优势，因此目前对药物发现非常感兴趣。这些优点包括：(i)使用较短的RNA避免干扰素诱导，（ii）使用更稳定的dsRNA，以及（iii） rnai介导的基因沉默具有催化作用，因此一个分子可以诱导许多目标mrna的切割。最近的报道吹捧了RNAi在基础和应用研究中的效用，旨在确定基因产物的功能和验证药物靶点。然而，由于核酸不容易穿过完整的活细胞膜，因此需要强大的技术将试剂输送到靶细胞中，以充分发挥rnai介导的基因沉默的潜力。我们开发了一种新的原型系统（称为LEAP(TM)），用于基于激光的细胞分析和操作。LEAP已被用于靶细胞的光注射试剂，最近用于实现RNAi和随后的高通量成像，以测量RNAi对许多单个细胞的影响。I期计划：I)评估光注射实现RNAi的新试剂，ii)优化光注射参数以实现高细胞活力和高效率的RNAi， iii)将结果扩展到具有高药物发现重要性的细胞和基因。这项工作将证明LEAP在光注射RNAi试剂的原位基因沉默方面的广泛应用。我们的初步数据已经吸引了几家大型制药/生物技术公司的注意，验证了所描述的方法的商业潜力。然后，第二阶段将提出开发特定的应用，如：i)高通量基因功能评估（例如，1536孔板的每孔中都有不同的基因），II)在同一细胞中同时沉默单独的基因，iii)沉默许多基因的整个途径，以及iv)专注于已知富含候选药物靶点的特定细胞过程。有了这些II期数据，基于LEAP和rnai介导的基因沉默的药物开发将进入III期商业化工作。

DESCRIPTION (provided by applicant): RNA interference (RNAi), a specific gene silencing method mediated by an intracellular enzyme complex using a dsRNA template, is of great current interest for drug discovery due to important advantages over older antisense methods. These advantages of include: (i) use of shorter RNA avoiding interferon induction, (ii) use of dsRNA which is more stable, and (iii) RNAi-mediated gene silencing is catalytic, so that one molecule can induce cleavage of many target mRNAs. Recent reports have touted the utility of RNAi for basic and applied research aimed at determining functions of gene products and validating drug targets. However, because nucleic acids do not readily pass through intact living cell membranes, robust techniques to deliver reagents into target cells are needed to fully realize the potential of RNAi-mediated gene silencing. We have developed a novel prototype system (called LEAP(TM)) for laser-based cell analysis and manipulation. LEAP has been used for optoinjection of reagents into target cells, most recently for achieving RNAi and subsequent high-throughput imaging to measure the RNAi effect on many individual cells. Phase I proposes to: i) evaluate new reagents for achieving RNAi with optoinjection, ii) optimize optoinjection parameters for high cell viability and high efficiency RNAi, and iii) extend results to cells and genes with high drug discovery importance. This work will demonstrate the broad utility of LEAP for in situ gene silencing by optoinjection of RNAi reagents. Our preliminary data has attracted the attention of several major pharmaceutical/biotechnology companies, validating the commercial potential of the approach described. Phase II would then propose to develop specific applications such as: i) high-throughput gene function evaluation (e.g., a different gene in each well of a 1536-well plate), ii) simultaneous silencing of separate genes in the same cells, iii) silencing whole pathways of many genes, and iv) focusing on specific cellular processes that are known to be rich in candidate drug targets. With such data from Phase II, a Phase III commercialization effort would be enabled for drug development based on LEAP and RNAi-mediated gene silencing.