Enhancing miRNA Therapeutics through Combinatorial Targeting and Vehicle Free Delivery

microRNAs（miRNAs）已经成为基因组的强大调控因子，通过共同努力， 鉴定它们功能并评估它们在体外和体内改变细胞生长的能力，其中一些已经获得青睐 作为潜在的治疗方法。虽然这些基于miRNA的方法可以彻底改变肿瘤的方式， 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，仍然存在的挑战是， 关于体内递送miRNA的问题必须解决。为了应对这些挑战，我们提出了两个 具体目标：一。为了通过基于miRNA的组合靶向增强miRNA治疗功效， 分子特征分析和II.开发和测试第二代载体用于递送未保护的miRNA。 我们广泛的初步证据支持这两个目标。我们最近从2019个miRNAs中鉴定出了10个 显著增强miR-34a的肿瘤抑制活性，miR-34a是第一个进入临床试验的miRNA， 并已开始鉴定这些miRNAs的直接靶点，以深入了解这些miRNAs的分子原因。 协同效应我们使用了一种新的方法，该方法依赖于将细胞miRNAs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。来自13个国家的测序数据 我们构建的文库将用于鉴定这些miRNAs的直接靶点，而不依赖于当前的 算法将对目标进行验证和评估，以确定其与开始 解释它们与miR-34a的协同作用。我们还建议评估在体内的疗效， 使用各种肺癌模型的组合对。尽管我们致力于了解 这些miRNAs与miR-34a协同作用，我们也建议使用这些数据来更好地理解miRNAs。 生物学在全球范围内因此，我们的数据将用于确定miRNAs如何与其靶点结合， 核苷酸分辨率，以及靶群体如何随着miRNA浓度而变化，这是 随着miRNA临床应用的增加，理解这一点非常重要。同时，我们将开发和测试一个 第二代miRNA递送载体，这是第一种完全递送miRNA的方法 不受保护在使用这种方法进行全身递送后，miRNA在肿瘤中特异性地积累， 并被致瘤细胞有效摄取，如靶基因阻遏所示， 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。 这将首次为无保护的miRNA递送提供证据。我们也将开始 打破了关于miRNA靶点鉴定的障碍，到目前为止，主要是使用 由于我们对miRNAs如何结合其靶标的理解存在差距，因此缺乏关键参数的算法。

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.