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

项目摘要/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人中就有1人患有哮喘。 尽管治疗方面的进步显著改善了疾病管理，但控制不佳的哮喘 仍然与严重的发病率和死亡率有关。当前改善哮喘控制的战略重点 关于减少炎症。GATA3是一种转录激活因子，参与T淋巴细胞分化和 信号，特别是在Th2亚型，并调节细胞因子的表达，如IL-4，IL-5和IL-2。 13，它们在导致哮喘的炎症中发挥主要作用。相应地，击倒GATA3 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)已经显示出 在动物模型和第一阶段临床试验中最有希望。DZS是一种典型的DNA寡核苷酸 催化降解特定的互补RNA序列。尽管可溶性DZS作为一种 治疗干预，通过质膜输送高电荷寡核苷酸，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 GATA3DNAzyme纳米颗粒结合，阐明了在肺中的稳定性和递送问题。 初步证据表明，~100DZS与一个14 nm的金粒子结合形成了一种络合物(DZNP)，该络合物 改善哮喘小鼠模型的呼吸道功能。重要的是，DzNPs使用的数量要低一个数量级 DZ剂量与其可溶性对应物进行比较。一个基本问题与DzNPs如何调解有关 提高了疗效。中心假设是DzNPs以不同的驻留细胞类型为目标 过度表达A类清道夫受体，这是Th2哮喘亚型的主要贡献者。为了测试 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 肺细胞株的DzNP摄取和GATA3基因敲除效率。我们的目标是了解DzNP如何 治疗不同于DZS，以及细胞靶点是否介导了疗效的提高。目标2将决定 DzNP是否充当DZ有效载荷的传递工具，或者DzNP结构是否具有功能性 GATA3调节的中间体。这将通过使用荧光寿命成像(FLIM)来实现 报告DZ分子的分子环境的显微镜探针。这位实习生将精通广场舞 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。这项拟议的研究将为使用一种新的基因调控方法提供洞察力。 合成生物材料。本研究将为纳米颗粒材料的未来发展提供基础 多种疾病的治疗策略。

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.