Inter and intra-cellular trafficking pathways for natural and therapeutic RNAs

天然和治疗性 RNA 的细胞间和细胞内运输途径

• 批准号: 8761471
• 负责人: ANASTASIA KHVOROVA
• 金额: $ 46.45万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-15 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8761471
• 关键词: Affinity; Antisense Oligonucleotides; Applications Grants; Biochemical; Biological; Biological Process; Biology; Caenorhabditis elegans; Cell Line; Cells; Charge; Chemicals; Chemistry; Clinic; Communication; Data; Development; Dissection; Drug Formulations; Engineering; Gene Silencing; Generations; Genes; Goals; Guanabenz; Human; Image; Imagery; Imaging technology; Interphase Cell; Kinetics; Knowledge; Label; Lead; Life; Lipids; Maps; Mass Spectrum Analysis; Mediating; MicroRNAs; Microscopy; Modeling; Molecular; Oligonucleotides; Pathway interactions; Procedures; Proteins; RNA; RNA Interference; Resolution; Signal Transduction; Small Interfering RNA; Small RNA; Solutions; Staging; Testing; Therapeutic; Tissues; Toxic effect; Validation; Vesicle; Whole Organism; analytical method; base; cellular imaging; clinical application; clinical efficacy; clinically relevant; design; extracellular; human Huntingtin protein; human disease; improved; next generation; novel; novel therapeutics; particle; phenylamil; protein complex; public health relevance; research study; small molecule; tool; trafficking; uptake

DESCRIPTION (provided by applicant): Highly inefficient transit of oligonucleotides from outside cells to the intracellular compartments where functional activity of oligonucleotides takes place is the most serious limitation to practical realization of a full potential of oligonucleotid-based therapies. Several classes of oligonucleotides therapeutics (ONT), including antisense oligonucleotides (ASO), hydrophobically modified siRNAs (hsiRNA), GalNAc conjugated siRNAs, and LNP formulated siRNAs have validated biological efficacy and are in clinic [1, 2]. In all cases, the fraction of oligonucleotides reaching the intended place of biological function is surprisingly low with the majority of molecules being trapped in wrong cellular compartments, resulting in low efficiency and clinically limiting toxicity. Concurrently, there is a significant nd rapidly expanding body of evidence demonstrating natural small RNA trafficking between cells as a means of intracellular communication. Given the extremely high efficacy of oligonucleotide-mediated gene silencing (25-100 molecules of Ago2 loaded siRNAs per cell [3]), exploiting the natural pathways of intracellular small RNA trafficking opens a possibility to dramatically improve the efficacy of ONTs. We hypothesize that exploiting natural, evolutionarily conserved mechanisms and pathways for trafficking of small RNAs across cellular boundaries is the way to fundamentally improve ONTs efficiency. The goal of this proposal is to elucidate the pathways and mechanisms involved in natural small RNA and ONTs trafficking and leverage this knowledge to increase the efficacy of oligonucleotide therapies. We have developed a unique set of experimental tools and analytical methods which enable us to systematically map and study endogenous small RNA as well as ONTs trafficking pathways. We have developed (1) a novel class of hydrophobic siRNAs (hsiRNAs), which efficiently enter cells and tissues; (2) an approach for visualization of native RNA trafficking through use of hsiRNA loaded exosomes as a model, (3) an approach for dissection of productive and non-productive ONTs trafficking using a panel of small molecules dramatically enhancing hsiRNAs efficacy by altering their cellular distribution; (4) a panel of four major classes of ONTs, which utilize different uptake mechanisms and silence a clinically relevant target huntingtin (htt), (5) a panel of engineered cell lines (GFP-fusions of trafficking markers) to study internalization pathways. Using these novel experimental tools along with TESM intracellular imaging, high resolution mass spectrometry and functional and biochemical analyses, we will identify the productive and non-productive cellular uptake mechanisms for both natural and synthetic oligonucleotides Completion of this project will result in (1) significant progress in our understanding of the natural mechanisms of extracellular uptake of small regulatory RNAs, (2) mapping uptake pathways for different classes of ONTs (3) development of the conceptual framework for generation of novel RNA chemistries relying on native uptake mechanisms. These findings will lead to a new generation of ONTs with dramatically improved clinical efficacy.

描述（由申请人提供）：寡核苷酸从细胞外转运至细胞内区室的效率极低，寡核苷酸的功能活性在细胞内区室中发挥作用 位置是实际实现基于寡核苷酸的疗法的全部潜力的最严重限制。几类寡核苷酸治疗剂 (ONT)，包括反义寡核苷酸 (ASO)、疏水修饰 siRNA (hsiRNA)、GalNAc 缀合 siRNA 和 LNP 配制的 siRNA 已验证生物功效并已投入临床 [1, 2]。在所有情况下，到达生物功能预期位置的寡核苷酸分数出人意料地低，大多数分子被困在错误的细胞区室中，导致效率低下和临床限制毒性。同时，大量且迅速扩大的证据表明细胞之间天然的小 RNA 运输是细胞内通讯的一种手段。鉴于寡核苷酸介导的基因沉默的极高功效（每个细胞加载 25-100 个 Ago2 分子的 siRNA [3]），利用细胞内小 RNA 运输的自然途径为显着提高 ONT 的功效提供了可能性。我们假设利用自然的、进化上保守的机制和途径来跨细胞边界运输小RNA是从根本上提高ONT效率的方法。该提案的目标是阐明天然小 RNA 和 ONT 贩运所涉及的途径和机制，并利用这些知识来提高寡核苷酸疗法的功效。我们开发了一套独特的实验工具和分析方法，使我们能够系统地绘制和研究内源性小 RNA 以及 ONT 贩运途径。我们开发了 (1) 一类新型疏水性 siRNA (hsiRNA)，可有效进入细胞和组织； (2) 一种通过使用 hsiRNA 负载的外泌体作为模型来可视化天然 RNA 运输的方法，(3) 一种使用一组小分子来剖析生产性和非生产性 ONT 运输的方法，通过改变 hsiRNA 的细胞分布来显着增强 hsiRNA 的功效； (4) 一组四个主要类别的 ONT，它们利用不同的摄取机制并沉默临床相关的目标亨廷顿蛋白 (htt)，(5) 一组工程细胞系（运输标记的 GFP 融合），用于研究内化途径。使用这些新颖的实验工具以及 TESM 细胞内成像、高分辨率质谱以及功能和生化分析，我们将确定天然和合成寡核苷酸的生产性和非生产性细胞摄取机制。该项目的完成将导致 (1) 我们对小调节 RNA 的细胞外摄取自然机制的理解取得重大进展，(2) 绘制图谱 不同类别 ONT 的摄取途径 (3) 开发依赖天然摄取机制生成新型 RNA 化学物质的概念框架。这些发现将导致新一代 ONT 的临床疗效显着提高。