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

• 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
• 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

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.

项目总结/摘要 哮喘是最常见的慢性呼吸系统疾病之一，估计在美国每13人中就有1人受到影响。 尽管治疗的进步显著改善了疾病管理，但控制不佳的哮喘仍是一个令人担忧的问题。 仍然与显著的发病率和死亡率相关。目前改善哮喘控制的策略重点是 减少炎症。GATA 3是一种转录激活因子，参与T淋巴细胞分化， 信号转导，特别是在Th 2亚型中，并调节细胞因子如IL-4、IL-5和IL-10的表达。 13，在哮喘的炎症中起主要作用。因此，GATA 3的击倒 表达是治疗具有Th 2内型的哮喘患者的有前景的策略。一系列反义 和RNAi技术已经过测试，在这些方法中，DNA酶（Dzs）已经显示出 在动物模型和1期临床试验中最有希望。Dz是典型的DNA寡核苷酸， 催化降解特定的互补RNA序列。尽管可溶性DZ作为一种 治疗性干预，递送高度带电的寡核苷酸穿过质膜，以及 防止核酸酶降解是主要的挑战。为了解决这些问题，我们建议开发 GATA 3 DNA酶纳米颗粒缀合物，其阐明了在肺中的稳定性和递送问题。 初步证据表明，将~100 Dzs与14 nm的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，DzNP使用低一个数量级的 Dz剂量与其可溶性对应物相比。一个基本问题涉及DzNP如何介导 提高功效。中心假设是DzNP差异性地靶向驻留细胞类型， 过表达A类清道夫受体，其是Th 2哮喘亚型的主要贡献者。测试 基于这一假设，我提出了以下具体目标：目标1将测量清道夫受体A的表达 在肺细胞系中的DzNP水平、DzNP摄取和GATA 3敲低效率。目标是了解DzNP如何 治疗与Dzs的治疗不同，以及细胞靶点是否介导改善的功效。目标2将决定 DzNP是否充当Dz有效载荷的运载工具，或者DzNP结构是否具有功能 GATA 3调节剂。这将通过采用荧光寿命成像（FLIM）来实现 报告Dz分子的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，包括纳米颗粒设计，表征，开发 和显微镜。这项研究将提供一种新的基因调控方法， 合成生物材料本研究为纳米材料的进一步发展奠定了基础 许多疾病的治疗策略。