Studies on RNAi Based Delivery in Vivo

描述(申请人提供)：基于RNAi的疗法正处于不同的研究阶段，用于治疗许多不同的疾病，包括病毒感染(例如，肝炎、艾滋病毒、流感、RSV)、癌症、高脂血症、亨廷顿病和老年性黄斑变性，仅举几例。我们之前的研究已经提供了关于组织中原代细胞的限速过程的重要信息，这些过程由于毒性而限制了RNAi的有效性。应用程序的第一部分旨在进一步剖析RNAi的分子局限性。我们建议进行研究，检查通过转录模板或直接双链传递的RNAi传递的局限性，同时仍然开发一种强大的RNAi传递系统，可以安全地将转基因表达降低3到4个数量级。这种水平的基因敲除对于正在考虑进行临床前研究和临床疗效的一些疾病可能是重要的。申请的第二部分涉及我们在检查哺乳动物RNAi途径时发现的新发现。首先，我们发现了有关microRNAs如何加载到RISC复合体中的新机制。其次，我们发现了3‘UTR中的miRNA靶标比mRNAs编码区的miRNA靶标更受青睐的生物学基础。第三个和第四个发现与识别两类新的小RNA有关。其中一组RNA可能参与了RNA指导的RNA转录，这一过程在无脊椎动物和植物中已知是强大的，但以前不被认为存在于哺乳动物中。第二类小RNA是由tRNA衍生而来。我们发现了两种不同类型的丰富的tRNA衍生的小RNA。我们已经在一定程度上确定了这些小RNA是如何从tRNA产生的。虽然我们已经确定这些RNAs定位于细胞质，但我们的初步数据表明，这些RNAs不是microRNA样的，尽管它们与特定的ArgAerte蛋白结合。我们假设这些RNAs可以作为缓冲区来调节可以被活性RISC复合体吸收的miRNAs的数量。我们建议进行研究，试图进一步阐明它们的功能。此外，我们发现，至少对于II型tRNA衍生的小RNA来说，有可能将它们与反义寡核苷酸结合使用，用于调控基因沉默，这一过程我们称为正义诱导反式沉默或SITS。我们建议进行研究，以确定将这种方法发展成真正的治疗方法的可能性。 公共卫生相关性：我们研究的最终目标是将小RNA用作治疗病毒感染、癌症和一些遗传疾病等疾病的药物。我们正在研究限制RNA干扰有效性的一些分子机制，以开发更强大的治疗方法。此外，我们还发现了其他类型的小RNA，它们的生物学功能尚不明确。我们正在研究这些RNA在基因调控中可能发挥的作用，并设计新的策略，将这些发展成为治疗疾病的新疗法。

DESCRIPTION (provided by applicant): RNAi-based therapeutics is in various stages of study for treating a number of different diseases, including viral infections (e.g., hepatitis, HIV, influenza, RSV), cancer, hyperlipidemia, Huntington's disease, and age-related macular degeneration, to name a few. Our previous studies have provided important information on the rate- limiting processes in primary cells in tissues that limit RNAi effectiveness due to toxicity. The first section of the application is designed to further dissect the molecular limitations of RNAi. We propose studies that examine the limitations of RNAi delivery via a transcriptional template or direct double-stranded delivery, while still developing a robust RNAi delivery system that can safely knock down transgene expression by 3 to 4 orders of magnitude. This level of knockdown will likely be important for a number of the diseases under consideration for preclinical study and clinical efficacy. The second part of the application deals with new discoveries that we uncovered while examining the RNAi pathway in mammals. First, we discovered new mechanisms on how microRNAs are loaded into the RISC complex. Second, we found the biologic basis for why miRNA targets in the 3'UTR are heavily favored over those in the coding regions of mRNAs. A third and forth discovery are related to identifying two new classes of small RNAs. One group of these RNAs may be involved in RNA-directed RNA transcription, a process known to be robust in invertebrates and plants but not previously believed to exist in mammals. The second class of small RNAs is derived from tRNAs. We discovered two different types of abundant tRNA- derived small RNAs. We have established in part how these small RNAs are generated from the tRNAs. While we have established that these RNAs are localized to the cytoplasm, our preliminary data suggest that these RNAs are not microRNA-like, even though they bind to specific argonaute proteins. We hypothesize that these RNAs serve as a buffer to regulate the number of miRNAs that can be taken up into an active RISC complex. We propose studies to attempt to further elucidate their function. Moreover, we have discovered that for at least the type II tRNA-derived small RNAs, there is the potential to use them in combination with an antisense oligonucleotide for regulated gene silencing in a process we call Sense-Induced-Trans Silencing or SITS. We propose studies to determine the likelihood of developing this approach into a bona-fide therapeutic. PUBLIC HEALTH RELEVANCE: The ultimate goal of our studies is to use small RNAs as therapeutics to treat diseases like viral infections, cancer, and some genetic disorders. We are studying some of the molecular mechanisms that limit RNA interference effectiveness in order to develop more robust treatment approaches. In addition, we have discovered other classes of small RNAs for which there is no defined biological function. We are studying the role of that these RNAs might play in gene regulation and designing new strategies to develop these into novel therapeutic approaches to treat disease.