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

项目摘要 治疗性核酸（TNAs）领域的迅速扩展导致了治疗性核酸（TNAs）领域的增加的紧迫性。 发展化学方法，可以扩大其临床应用。在过去的三年里，美国 美国食品和药物管理局已经批准了两种基于TNA的药物，用于治疗以前几乎没有 治疗的选择。尽管最近取得了这些成就，但许多涉及TNA的临床试验仍导致终止妊娠。 由于有限的功效，这一结果通常归因于由于高极性和带电的 TNA的特征限制了细胞胞质溶胶中mRNA的可及性。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，其有效递送的中心瓶颈是逃离内体区室并获得 进入细胞质我的研究计划的中心愿景是解决这些挑战与 内体逃逸和TNA在分子水平上递送到胞质溶胶。我们的目标是应对这些挑战 通过合成核酸表面活性剂缀合物，我们最近已经证明可以成功靶向 用于体外基因沉默的mRNA。由于它们的设计是化学可调的，我们的目标是系统地评估其作用， 表面活性剂，因为它涉及到内体逃逸的程度，通过评估疏水的重要性， 特征、净电荷和缀合物的大小。我们还将开发一类新的荧光表面活性剂 可用于监测表面活性剂缀合物的递送和稳定性的探针 通过细胞贩运，从而帮助我们量化的影响变化的化学性质的 缀合物对胞质递送的影响。结合这些研究，我们将确定 化学修饰核酸表面活性剂缀合物对体外基因敲低功效的影响， 导致mRNA切割的DNA酶。通过控制单个DNA酶-表面活性剂的分子设计， 结合物，我们可以更好地了解其进入细胞的机制和导致细胞溶质的性质。 access.通过成功实现我们的计划，我们不仅将有助于我们对 这些性质是核酸成功进入细胞胞质溶胶所必需的，而且还设计了 该平台将具有适用于未来转化应用的即时治疗价值。

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.