Elucidation of DNA Enzyme Nanoparticles in Regulating Gene Expression in the Lung

DNA 酶纳米颗粒调节肺基因表达的阐明

基本信息

批准号：
9401923
负责人：
Nusaiba Baker
金额：
$ 4.9万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-22 至 2020-08-21
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9401923
关键词：
Address Adrenal Cortex Hormones Affect Animal Model Asthma Biocompatible Materials Biological Biopsy Breathing CD4 Positive T Lymphocytes Catalytic DNA Cell Line Cell membrane Cells Characteristics Charge Chronic Chronic Disease Complementary RNA Complex Cytoplasm DNA Data Development Disease Disease Management Dose Engineering Environment Enzymes Epithelial Cells Extrinsic asthma Fibroblasts Foundations Future GATA3 gene Gene Expression Gene Expression Regulation Goals Gold Half-Life Helper-Inducer T-Lymphocyte Human Immobilization Individual Inflammation Inflammatory Response Interferon Type II Interleukin-13 Interleukin-4 Interleukin-5 Kinetics Location Lung Maps Measures Mediating Messenger RNA Methods Microscopy Molecular Morbidity - disease rate Multienzyme Complexes Natural Immunity Oligonucleotides Oral Patients Pharmacologic Substance Phase Phase I Clinical Trials Play Public Health RNA Interference RNA Sequences Regulation Reporting Research Respiration Disorders Rodent Role SR-A proteins Serum Signal Transduction Small Interfering RNA Smooth Muscle Myocytes T-Lymphocyte Techniques Technology Testing Therapeutic Therapeutic Intervention Transcription Coactivator Up-Regulation Zinc Fingers adaptive immunity airway epithelium airway hyperresponsiveness asthmatic asthmatic patient attenuation base cell type cytokine design eosinophil experimental study fluorescence lifetime imaging improved insight knock-down macrophage mast cell mortality mouse model nanoparticle novel novel therapeutics nuclease overexpression particle prevent response scaffold scavenger receptor success targeted agent uptake vector

项目摘要

PROJECT SUMMARY/ABSTRACT Asthma is one of the most common chronic respiratory disorders, estimated to affect 1 in 13 people in the U.S. Although advances in treatment have significantly improved disease management, poorly controlled asthma is still associated with significant morbidity and mortality. The current strategy to improve asthma control focuses on reducing inflammation. GATA3, a transcriptional activator, is involved in T lymphocyte differentiation and signaling, particularly in the Th2 subtype, and regulates the expression of cytokines such as IL-4, IL-5, and IL- 13, which play major roles in the inflammation responsible for asthma. Accordingly, knock down of GATA3 expression is a promising strategy for treating asthmatic patients with the Th2 endotype. A range of antisense and RNAi technologies have been tested, and among these approaches, DNA enzymes (Dzs) have shown the greatest promise in animal models and Phase 1 clinical trials. Dzs are canonical DNA oligonucleotides that catalytically degrade a specific complementary RNA sequence. Despite the success of soluble Dzs as a therapeutic intervention, delivering highly charged oligonucleotides across the plasma membrane, and preventing nuclease degradation are major challenges. To address these problems, we propose developing GATA3 DNAzyme nanoparticle conjugates that elucidate the stability and delivery issues in the lung. Preliminary evidence shows that conjugating ~100 Dzs to a 14-nm gold particle forms a complex (DzNP) that improves airway function in mouse models of asthma. Importantly, DzNPs use one order of magnitude lower Dz dose compared to their soluble counterparts. A fundamental question pertains to how DzNPs mediate improved efficacy. The central hypothesis is that DzNPs differentially target resident cell types that overexpress class A scavenger receptors, which are primary contributors to the Th2 asthma subtype. To test this hypothesis, I propose the following specific aims: Aim 1 will measure scavenger receptor A expression levels, DzNP uptake, and GATA3 knockdown efficiency in lung cell lines. The goal is understanding how DzNP treatment differs from that of Dzs and whether the cell targets mediate improved efficacy. Aim 2 will determine whether the DzNP acts as a delivery vehicle for Dz payloads, or whether the DzNP construct is the functional agent mediating GATA3 regulation. This will be achieved by employing fluorescence lifetime imaging (FLIM) microscopy probes that report the molecular environment of the Dz molecule. The trainee will master a wide range of engineering and biological techniques, including nanoparticle design, characterization, development and microscopy. The proposed research will provide insight into a novel method of gene regulation using a synthetic biomaterial. This research will provide a foundation for future development of nanoparticle-based therapeutic strategies for numerous diseases.

项目概要/摘要哮喘是最常见的慢性呼吸系统疾病之一，据估计，美国每 13 个人中就有 1 人患有哮喘。尽管治疗的进步显着改善了疾病管理，但哮喘控制不佳仍与显着的发病率和死亡率相关。当前改善哮喘控制的策略重点是关于减少炎症。 GATA3 是一种转录激活剂，参与 T 淋巴细胞分化和信号传导，特别是在 Th2 亚型中，并调节细胞因子的表达，例如 IL-4、IL-5 和 IL- 13，它在导致哮喘的炎症中起主要作用。因此，GATA3的敲低表达是治疗 Th2 内型哮喘患者的一种有前景的策略。一系列反义和 RNAi 技术已经过测试，在这些方法中，DNA 酶 (Dzs) 已显示出在动物模型和一期临床试验中最有希望。 Dzs 是典型的 DNA 寡核苷酸，催化降解特定的互补RNA序列。尽管可溶 Dzs 取得了成功治疗干预，跨质膜传递高电荷寡核苷酸，以及防止核酸酶降解是主要挑战。为了解决这些问题，我们建议开发 GATA3 DNAzyme 纳米颗粒缀合物阐明了肺部的稳定性和递送问题。初步证据表明，将约 100 Dzs 与 14 nm 金颗粒结合形成复合物 (DzNP)，改善哮喘小鼠模型的气道功能。重要的是，DzNPs 使用的数量级要低一个数量级 Dz 剂量与其可溶性对应物相比。一个基本问题涉及 DzNPs 如何调节提高疗效。中心假设是 DzNPs 不同地靶向常驻细胞类型，过度表达 A 类清道夫受体，这是 Th2 哮喘亚型的主要贡献者。测试根据这个假设，我提出以下具体目标：目标 1 将测量清道夫受体 A 的表达肺细胞系中的水平、DzNP 摄取和 GATA3 敲低效率。目标是了解 DzNP 如何治疗与 Dzs 的不同以及细胞靶点是否介导疗效的改善。目标 2 将决定 DzNP 是否充当 Dz 有效负载的传递载体，或者 DzNP 构造是否是功能性的介导 GATA3 调节的代理。这将通过采用荧光寿命成像 (FLIM) 来实现报告 Dz 分子分子环境的显微镜探针。受训者将掌握广泛的一系列工程和生物技术，包括纳米粒子设计、表征、开发和显微镜。拟议的研究将深入了解一种利用基因调控的新方法合成生物材料。该研究将为纳米粒子的未来发展奠定基础许多疾病的治疗策略。