Maximizing siRNA Function through Mechanism-based Sequence and Vehicle Design

描述（由申请人提供）：RNA干扰（RNAi）是真核细胞中通过引入互补双链RNA（dsRNA）降低靶mRNA表达的内源性途径。RNAi的发现为分析生物网络和鉴定治疗靶点提供了手段。由于免疫原性的原因，哺乳动物细胞中的RNAi由短干扰RNA（siRNA）启动。尽管迄今为止对RNAi途径和siRNA的使用进行了深入的研究，但鉴定最具活性的序列并将这些序列有效递送至感兴趣的细胞仍然是重大的挑战。本文提出通过分析RNAi途径中的多个步骤来解决siRNA选择和递送的这些挑战。拟议的工作将集中在优化siRNA功能的两种方法。首先，将表征siRNA与重要RNAi途径蛋白的结合相互作用。已知每种蛋白质都是RNAi途径的核心，但它们对沉默的贡献尚未完全了解。其次，将定量分析siRNA与由化学多样性寡聚和聚合纳米颗粒构建的递送载体的相互作用，以确定促进复合物形成、保护siRNA免于降解以及siRNA在进入细胞后释放的那些结构特征。要研究的聚合物纳米粒子很容易被修改，以提供一种方法，创建，与精致的控制，一个不同的数组的车辆，我们将使用测试变量，如胺密度，聚乙二醇改性，亲水性，结合协同性。该研究的总体目标是通过操纵siRNA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的治疗剂必须被设计为在其复杂的作用机制中的各种步骤中具有最佳功能。拟议的工作将解决这些重要步骤中的两个，为基于RNAi的高活性，高特异性疗法的设计提供指导。

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.