Maximizing siRNA Function through Mechanism-based Sequence and Vehicle Design

描述(申请人提供)：RNA干扰(RNAi)是真核细胞中的一种内源性途径，通过引入互补双链RNA(DsRNA)来减少目标mRNA的表达。RNAi的发现为分析生物网络和识别治疗靶点提供了一种手段。由于免疫原性的原因，哺乳动物细胞中的RNAi是由短干扰RNA(SiRNAs)启动的。尽管到目前为止对RNAi途径和siRNAs的使用进行了密集的研究，但识别最活跃的序列并将这些序列有效地传递到感兴趣的细胞仍然是一个巨大的挑战。本文提出通过分析RNAi途径中的多个步骤来解决siRNA选择和传递的这些挑战。建议的工作将集中在两种优化siRNA功能的方法上。首先，将表征siRNAs与重要的RNAi途径蛋白的结合作用。已知每种蛋白质都是RNAi途径的中心，但它们对沉默的作用尚不完全清楚。其次，将对siRNAs与由化学上不同的寡聚和聚合物纳米颗粒构建的输送载体的相互作用进行定量分析，以确定那些鼓励复合体形成、保护siRNAs免受降解以及siRNAs进入细胞后释放的结构特征。要研究的聚合物纳米颗粒很容易被修饰，以提供一种方法，通过精确的控制，创建一系列不同的载体，我们将使用这些载体来测试变量，如胺密度、聚乙二醇改性、亲水性和结合协作性。这项研究的总体目标是通过操纵siRNA的结构和序列以及设计具有最佳化学和物理特性的载体来设计具有最大功能的siRNA。 公共卫生相关性：RNA干扰(RNAi)是一种阻断特定蛋白质表达的技术，有可能成为一种重要的治疗策略。为了以最大的安全性和活性实现这一潜力，基于RNAi的疗法必须被设计为在其复杂的作用机制中的各种步骤具有最佳功能。拟议的工作将涉及这些重要步骤中的两个，以提供基于RNAi的高活性、高特异性疗法的设计指南。

DESCRIPTION (provided by applicant): RNA interference (RNAi) is an endogenous pathway in eukaryotic cells for reducing the expression of a target mRNA through the introduction of complementary double-stranded RNA (dsRNA). The discovery of RNAi has provided a means for analysis of biological networks and identification of therapeutic targets. For reasons of immunogenicity, RNAi in mammalian cells is initiated by short interfering RNAs (siRNAs). Despite the intense study to date on the RNAi pathway and the use of siRNAs, the identification of the most active sequences and efficient delivery of those sequences to the cells of interest remain significant challenges. It is proposed here to address these challenges of siRNA selection and delivery through analysis of multiple steps in the RNAi pathway. The proposed work will focus on two approaches to optimizing siRNA function. First, the binding interactions of siRNAs with important RNAi pathway proteins, will be characterized. Each of the proteins is known to be central to the RNAi pathway, but their contributions to silencing are not fully-understood. Second, the interactions of siRNAs with delivery vehicles built from chemically-diverse oligomeric and polymeric nanoparticles will be quantitatively analyzed to determine those structural features that encourage complex formation, protection of the siRNAs from degradation, and release of siRNAs upon entry into the cell. The polymeric nanoparticles to be studied are readily modified to provide a means of creating, with exquisite control, a diverse array of vehicles that we will use to test variables such as amine density, polyethylene glycol modification, hydrophilicity, and binding cooperativity. The overall goal of the proposed research is to design siRNAs with maximal function through manipulation of the siRNA structure and sequence and the design of vehicles with optimal chemical and physical characteristics. PUBLIC HEALTH RELEVANCE: RNA interference (RNAi), a technique for blocking the expression of a specific protein, has the potential to be an important therapeutic strategy. To realize this potential with maximum safety and activity, RNAi-based therapeutics must be designed to have optimal function for a variety of steps in their complex mechanism of action. The proposed work will address two of these important steps to provide guidelines for the design of highly-active, highly-specific therapeutics based on RNAi.