Maximizing siRNA Function through Mechanism-based Sequence and Vehicle Design

通过基于机制的序列和载体设计最大化 siRNA 功能

基本信息

批准号：
8535167
负责人：
Stephen Patrick Walton
金额：
$ 22.3万
依托单位：
MICHIGAN STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-01 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8535167
关键词：
Abbreviations Address Affinity Alzheimer&apos s Disease Amines Base Pairing Base Sequence Binding Biological Biological Process Cataloging Catalogs Cell Culture Techniques Cell membrane Cells Characteristics Charge Chemical Engineering Chemicals Chemistry Cholesterol Cleaved cell Clinical Complex Computing Methodologies Cytoplasm DNA Development Diabetes Mellitus Disease Dissociation Double-Stranded RNA Drug Delivery Systems EMSA Electrophoretic Mobility Shift Assay Electrostatics Enzymes Eukaryotic Cell Foundations Future Gel Chromatography Gene Expression Genes Goals Guidelines Human Knowledge Laboratories Ligands Lysine MAPK8 gene Malignant Neoplasms Mammalian Cell Measures Mediating Messenger RNA MicroRNAs Modification Molecular Nucleic Acids Nucleotides Organism Pathway interactions Pharmaceutical Preparations Polyethylene Glycols Polyethyleneimine Polymerase Chain Reaction Polymers Property Proteins Qualifying RNA RNA Degradation RNA Interference RNA Interference Pathway RNA-Binding Proteins RNA-Induced Silencing Complex RNA-Protein Interaction Research Reverse Transcription Ribonucleases Safety Science Small Interfering RNA Structure Techniques Technology Testing Therapeutic Toxic effect Transmission Electron Microscopy Viral Work analog base cytotoxicity density design enhanced green fluorescent protein experience graduate student human DICER1 protein hydrophilicity immunogenicity interest intermolecular interaction light scattering lipofection meetings nanoparticle nucleic acid structure professor protein kinase R public health relevance receptor research study skills stress-activated protein kinase 1 therapeutic target

项目摘要

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.

描述（由申请人提供）：RNA干扰（RNAi）是真核细胞中的一种内源途径，通过引入互补的双链RNA（dsRNA）来减少靶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。出于免疫原性的原因，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，但识别最活跃的序列并将这些序列有效地递送至感兴趣的细胞仍然是重大挑战。本文建议通过分析 RNAi 途径中的多个步骤来解决 siRNA 选择和递送的这些挑战。拟议的工作将重点关注两种优化 siRNA 功能的方法。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 与由化学多样化的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 免遭降解以及进入细胞后 siRNA 释放的结构特征。待研究的聚合物纳米粒子很容易进行修饰，以提供一种通过精确控制创建多种载体的方法，我们将使用这些载体来测试胺密度、聚乙二醇修饰、亲水性和结合协同性等变量。本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 的疗法必须设计为在其复杂作用机制的各个步骤中具有最佳功能。拟议的工作将解决其中两个重要步骤，为基于 RNAi 的高活性、高特异性疗法的设计提供指导。