CCR RNAi Initiative: Validation of siRNAs Against Cancer

CCR RNAi 计划：验证 siRNA 抗癌作用

• 批准号: 7338744
• 负责人: natasha caplen
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7338744
• 关键词: CCR; RNAi; Initiative; Validation; siRNAs

To access large-scale synthetic siRNA resources Gene Silencing Section, Genetics Branch, has developed a research collaboration agreement with Qiagen Inc. To date Qiagen has designed and synthesized siRNAs corresponding to approximately 400 genes. To make full use of these resources we have developed a high throughput automated synthetic siRNA-lipid "reverse transfection" protocol in a 96 well plate format (BioRobot 8000, Qiagen Inc.). To date we have fully assayed the knockdown mediated by 258 siRNAs corresponding to 129 human genes. Approximately 70% of these siRNAs show a 50% decrease in the steady state levels of the expression of the gene under study. We have conducted extensive study of the reasons why some siRNAs fail to silence and have established the single nucleotide polymorphisms, errors and changes in the consensus transcript and genomic sequences used for the design of siRNAs and differential expression of transcript variants can all contribute to the seeming failure of an siRNA to mediate RNAi. We have enabled CCR investigators to access the confidential RNAi analysis data for 100 human genes, and this has resulted in the execution of over 30 intramural material transfer agreements and the transfer of appropriate information to CCR investigators. The following describes one example of the impact of this validation process. Using synthetic siRNAS corresponding to the enzyme asparagine synthetase we had previously validated Dr. John Weinstein's group (LMP, CCR) were able to rapidly assess a functional relationship between ASNS expression and the activity of the enzyme drug L-asparaginase (L-ASP). This work has lead to the filing of a patent application and a manuscript describing this work in detail is In Press. This study showed that treatment of cells with siRNAs targeted against ASNS reduces ASNS expression and potentiates the growth inhibitory activity of L-asparaginase (L-ASP), a FDA approved drug used for the treatment of chronic lymphocytic leukemia. This data suggests that L-ASP treatment could be applied to other cancers and suggest that ASNS could be used as a biomarker for the clinical effectiveness of L-ASP. This study presents a paradigm for the use of RNAi analysis to further pharmocogenomic studies.To go further in our use of RNAi analysis we have needed to establish large-scale RNAi screening methods. Our early consideration of siRNA mediated RNAi screens using arrayed siRNAs suggested that this format is associated with a number of practical limitations. Such limitations include the potential need for extensive automated liquid handling and/or the requirement for large amounts of transfection and assay reagents, which can be cost-prohibitive. In addition, some assay platforms are incompatible with large-scale analysis because of the limited number of samples that can be assayed at any one time. Many of these assays can also be labor intensive. Furthermore, the number of targets exhibiting a given assayed phenotype (i.e. "hits") is likely to correspond to only a small fraction of the total targets evaluated in larger unbiased screens, retrospectively making the majority of such screens superfluous. While some of these issues have been addressed for shRNAs no studies have examined this in relation to synthetic siRNAs. To address this we developed a novel strategy using multiplexes of synthetic siRNAs corresponding to multiple gene targets in order to streamline siRNA RNAi screens. We have established that multiplexed siRNAs can silence at least six genes to the same degree as when the genes were targeted individually. We then generated libraries of siRNAs containing randomly multiplexed siRNAs and used them in a screen of cell viability. Using this strategy, several gene targets that influenced the viability of a breast cancer cell line were identified. Included were known or putative anti-tumor molecular targets.

为了获得大规模的合成siRNA资源，基因沉默科，遗传学分支，已经与Qiagen Inc.达成了一项研究合作协议。到目前为止，Qiagen已经设计并合成了对应于大约400个基因的siRNA。为了充分利用这些资源，我们开发了96孔板形式的高通量自动化合成siRNA-脂质“反向转染”方案（BioRobot 8000，Qiagen Inc.）。到目前为止，我们已经完全测定了由对应于129个人类基因的258个siRNA介导的敲低。这些siRNA中约70%显示出所研究基因表达的稳态水平降低50%。我们已经对一些siRNA不能沉默的原因进行了广泛的研究，并且已经确定了用于设计siRNA的共有转录物和基因组序列中的单核苷酸多态性、错误和变化以及转录物变体的差异表达都可能导致siRNA介导RNAi的失败。我们使CCR研究人员能够访问100个人类基因的机密RNAi分析数据，这导致了30多个内部材料转让协议的执行，并将适当的信息转移给CCR研究人员。以下描述了此验证过程影响的一个示例。使用与天冬酰胺合成酶相对应的合成siRNAS，我们先前已经验证了John Weinstein博士的小组（LMP，CCR）能够快速评估ASNS表达与酶药物L-天冬酰胺酶（L-ASP）活性之间的功能关系。这项工作已导致提交专利申请和手稿详细描述这项工作是在新闻。这项研究表明，用靶向ASNS的siRNA处理细胞可降低ASNS表达，并增强L-天冬酰胺酶（L-ASP）的生长抑制活性，L-ASP是FDA批准的用于治疗慢性淋巴细胞白血病的药物。这些数据表明，L-ASP治疗可应用于其他癌症，并表明ASNS可用作L-ASP临床有效性的生物标志物。本研究为RNAi分析在药物基因组学研究中的应用提供了一个范例，为了进一步应用RNAi分析，我们需要建立大规模的RNAi筛选方法。我们早期对使用阵列siRNA进行siRNA介导的RNAi筛选的考虑表明，这种形式与许多实际限制有关。这样的限制包括对广泛的自动化液体处理的潜在需求和/或对大量转染和测定试剂的需求，这可能是成本过高的。此外，一些测定平台与大规模分析不兼容，因为在任何一个时间可以测定的样品数量有限。这些测定中的许多也可能是劳动密集型的。此外，表现出给定测定表型（即“命中”）的靶的数量可能仅对应于在较大的无偏筛选中评价的总靶的一小部分，回顾性地使得大多数这样的筛选是多余的。虽然这些问题中的一些已经针对shRNA得到了解决，但是没有研究针对合成siRNA对此进行了研究。为了解决这个问题，我们开发了一种新的策略，使用对应于多个基因靶点的合成SiRNA多重化，以简化SiRNA RNAi筛选。我们已经确定，多重siRNA可以沉默至少六个基因，其程度与单独靶向基因时相同。然后，我们生成了含有随机多重siRNA的siRNA文库，并将其用于细胞活力的筛选。使用这种策略，确定了影响乳腺癌细胞系活力的几个基因靶点。包括已知或推定的抗肿瘤分子靶点。