Achieving enhanced cytosolic delivery and greater efficacy of therapeutic nucleic acids using DNA-surfactant conjugates

项目摘要 治疗性核酸(TNA)领域的迅速扩大导致了在 开发可扩大其临床应用的化学方法。仅在过去的3年里，美国 美国食品和药物管理局批准了两种基于TNA的药物，用于治疗以前几乎没有什么疗效的疾病 治疗的选择。尽管最近取得了这些成就，但许多涉及TNA的临床试验都导致了终止 由于疗效有限，这一结果通常被归因于由于高极性和带电而导致的交付不良 限制细胞胞浆中信使核糖核酸的可及性的TNAs的特征。许多平台提供核酸 使用基于纳米颗粒的方法，通过内吞作用机制内化到细胞中。 因此，它们有效传递的一个中心瓶颈是逃离内体隔室并获得 进入细胞质的途径。我的研究计划的中心愿景是解决与以下方面相关的挑战 在分子水平上，胞体逃逸和TNA递送到胞浆。我们的目标是应对这些挑战 通过合成我们最近显示的核酸表面活性物质偶联物，可以成功地靶向 基因沉默的信使核糖核酸。由于他们的设计是化学可调的，我们的目标是系统地评估这种作用 通过评估疏水性的重要性来评价表面活性物质与内质粒逃逸程度的关系 偶联物的性质、净电荷和大小。我们还将开发一种新的荧光表面活性剂 可用于监测表面活性物质偶联物的输送和稳定性的探针 通过细胞运输，从而帮助我们量化影响的化学性质的变化 偶联物对胞液输送有影响。结合这些研究，我们将确定 化学修饰核酸表面活性物质结合物对体外基因敲除效果的影响 导致信使核糖核酸切割的脱氧核酶。通过控制单个DNA酶-表面活性物质的分子设计 我们可以更好地了解它们进入细胞的机制和导致胞浆的性质 进入。通过成功地实现我们的计划，我们不仅将有助于我们对 核酸成功进入细胞胞浆所必需的特性，但也设计了 一个平台，将具有立竿见影的治疗价值，适合未来的翻译应用。

Project Summary The rapidly expanding field of therapeutic nucleic acids (TNAs) has led to an increased urgency in the development of chemical approaches that can broaden their clinical application. In just the last 3 years the US Food and Drug Administration has approved two TNA based drugs that treat diseases that previously had little option for treatment. Despite these recent achievements, many clinical trials involving TNAs result in termination due to limited efficacy, a result that is often attributed to poor delivery owing to the highly polar and charged character of TNAs that limits accessibility to mRNA in the cytosol of cells. Many platforms deliver nucleic acids using nanoparticle-based approaches that are internalized into cells through endocytosis mechanisms. Therefore, a central bottleneck to their effective delivery is escaping endosomal compartments and gaining access to the cytosol. The central vision of my research program is to address these challenges associated with endosomal escape and TNA delivery to the cytosol at the molecular level. We aim to address these challenges through the synthesis of nucleic acid surfactant conjugates that we have recently shown can successfully target mRNA for gene silencing in vitro. As their design is chemically tunable, we aim to systematically assess the role of the surfactant as it relates to the extent of endosomal escape by evaluating the importance of hydrophobic character, net charge and size of the conjugates. We will also develop a new class of fluorescent surfactant probes which can be used to monitored the delivery and stability of the surfactant conjugates as they are trafficked through cells, thereby helping us to quantify the effects changes in the chemical character of the conjugates have on cytosolic delivery. In conjunction with these studies we will determine the net effect of chemically modifying the nucleic acid surfactant conjugates on the efficacy of gene knockdown in vitro using DNAzymes that result in mRNA cleavage. By controlling the molecular design of individual DNAzyme-surfactant conjugates we can better understand their mechanism of cellular entry and the properties that lead to cytosolic access. Through successful realization of our program we will not only contribute to our understanding of the properties that are necessary for nucleic acids to successfully gain access to the cytosol of cells but also design a platform that will have immediate therapeutic value suitable for future translational applications.