Enhancing miRNA Therapeutics through Combinatorial Targeting and Vehicle Free Delivery

MicroRNA (miRNA) 已成为基因组的强大调节剂，并且通过共同努力 鉴定它们的功能并评估它们在体外和体内改变细胞生长的能力，其中一些已获得青睐 作为潜在的治疗方法。尽管这些基于 miRNA 的方法可以彻底改变肿瘤的治疗方式 通过诊断和治疗，我们对 miRNA 生物学的功能和分子方面的理解是 仍然不完整。此外，为了将 miRNA 生物学与临床应用联系起来，仍然存在挑战 必须解决 miRNA 的体内递送问题。为了应对这些挑战，我们提出了两个建议 具体目标： I. 通过基于 miRNA 的组合靶向和治疗来增强 miRNA 的治疗功效 分子谱分析和 II.开发和测试用于传递未受保护的 miRNA 的第二代载体。 我们广泛的初步证据支持这两个目标。我们最近从 2,019 个 miRNA 中鉴定出 10 个 显着增强第一个进入临床试验的miRNA miR-34a的肿瘤抑制活性， 并已开始确定这些 miRNA 的直接靶标，以深入了解造成这种情况的分子原因 协同效应。我们使用了一种新颖的方法，该方法依赖于将细胞 miRNA 直接连接到它们的 相关的 RNA 靶标，然后对 RNA 杂交体进行深度测序。测序数据来自13 我们构建的文库将用于识别这些 miRNA 的直接目标，独立于当前的 算法。目标将被验证和评估与其相关的途径，这些途径将开始 解释它们与 miR-34a 的协同作用。我们还建议评估其体内功效 使用各种肺癌模型的组合对。尽管我们致力于了解如何 这些 miRNA 与 miR-34a 合作，我们还建议使用这些数据来更好地理解 miRNA 全球水平的生物学。因此，我们的数据将用于确定 miRNA 如何与其靶标相关联 核苷酸分辨率，以及目标群体如何随 miRNA 浓度而变化，即 随着 miRNA 临床效用的增加，理解这一点极其重要。与此同时，我们将开发和测试 第二代 miRNA 递送载体，这是一种完全递送 miRNA 的一流方法 不受保护。使用这种方法进行全身递送后，miRNA 会在肿瘤中特异性积累 并被致瘤细胞有效吸收，如靶基因抑制所示，没有明显的影响 毒性。总的来说，从这项工作中获得的数据将验证组合 miRNA 的体内功效 疗法，并将首次为未受保护的 miRNA 传递提供证据。我们也将开始 打破 miRNA 靶点识别的障碍，到目前为止，大多数方法都是使用 由于我们对 miRNA 如何结合其靶标的理解存在差距，这些算法缺乏关键参数。

MicroRNAs (miRNAs) have emerged as powerful regulators of the genome and, through concerted efforts to identify their function and evaluate their ability to alter cell growth in vitro and in vivo, some have gained favor as potential therapeutics. Although these miRNA-based approaches can revolutionize the way that tumors are diagnosed and treated, our understanding of the functional and the molecular aspects of miRNA biology are still incomplete. Moreover, in order to bridge miRNA biology with clinical utility, the challenges that still remain with regard to in vivo delivery of miRNAs must be tackled. To address these challenges we propose two Specific Aims: I. To enhance miRNA therapeutic efficacy through combinatorial miRNA-based targeting and molecular profiling and II. To develop and test second-generation vehicles for delivery of unprotected miRNAs. Our extensive preliminary evidence supports both Aims. We recently identified 10 miRNAs out of 2,019 that significantly enhance the tumor suppressive activity of miR-34a, the first miRNA to enter into clinical trial, and have begun to identify the direct targets of these miRNAs to gain insight into the molecular reason for the cooperative effect. We used a novel method that relies on ligating the cellular miRNAs directly to their associated RNA target followed by deep sequencing of the RNA hybrids. The sequencing data from 13 libraries that we constructed will be used to identify the direct targets of these miRNAs, independent of current algorithms. Targets will be validated and evaluated for pathways that they associate with that will begin to explain their cooperative effect with miR-34a. We also propose to evaluate the in vivo efficacy of the combinatorial pairs using various models of lung cancer. Although we are committed to understanding how these miRNAs are cooperating with miR-34a we also propose to use this data to better understand miRNA biology at a global level. Thus, our data will be used to determine how miRNAs associate with their targets at nucleotide resolution, and how the target population changes with regard to miRNA concentration, which is extremely important to understand as miRNA clinical utility increases. In parallel we will develop and test a second-generation miRNA delivery vehicle, which is a first-in-class method for delivering miRNAs completely unprotected. Following systemic delivery using this method, the miRNA accumulates specifically in the tumor and is efficiently taken up by the tumorigenic cells as indicated by target gene repression with no obvious toxicity. Collectively, the data obtained from this work will validate in vivo efficacy for combinatorial miRNA therapeutics, and for the first time will provide evidence for unprotected miRNA delivery. We will also begin to break down the barriers regarding miRNA target identification that until now has been mostly approached using algorithms that lack critical parameters due to a gap in our understanding of how miRNAs bind their targets.