Studies on RNAi Based Delivery in Vivo

描述（由申请人提供）：基于RNA干扰的治疗方法正处于治疗多种不同疾病的不同研究阶段，包括病毒感染（例如，肝炎、HIV、流感、RSV）、癌症、高脂血症、亨廷顿氏病和年龄相关性黄斑变性，仅举几例。我们先前的研究提供了关于组织中原代细胞中由于毒性而限制RNAi有效性的限速过程的重要信息。该申请的第一部分旨在进一步剖析RNAi的分子限制。我们提出研究，检查通过转录模板或直接双链递送的RNAi递送的局限性，同时仍然开发一个强大的RNAi递送系统，可以安全地敲低转基因表达3至4个数量级。这种敲低水平对于临床前研究和临床疗效所考虑的许多疾病可能是重要的。 申请的第二部分涉及我们在研究哺乳动物中的RNAi途径时发现的新发现。首先，我们发现了microRNA如何加载到RISC复合物中的新机制。其次，我们发现了为什么3'UTR中的miRNA靶点比mRNA编码区中的靶点更受青睐的生物学基础。第三个和第四个发现与鉴定两类新的小RNA有关。这些RNA中的一组可能参与RNA指导的RNA转录，这一过程已知在无脊椎动物和植物中是稳健的，但以前认为不存在于哺乳动物中。第二类小RNA来自tRNA。我们发现了两种不同类型的丰富的tRNA衍生的小RNA。我们已经部分确定了这些小RNA是如何从tRNA中产生的。虽然我们已经确定这些RNA定位于细胞质，但我们的初步数据表明，这些RNA不是microRNA样的，即使它们与特定的argonaute蛋白结合。我们假设这些RNA作为缓冲液来调节可以被吸收到活性RISC复合物中的miRNA的数量。我们建议研究，试图进一步阐明其功能。此外，我们已经发现，至少对于II型tRNA衍生的小RNA，有可能将它们与反义寡核苷酸组合使用，以在我们称为有义诱导的反式沉默或SITS的过程中调节基因沉默。我们建议进行研究，以确定将这种方法发展成为真正的治疗方法的可能性。

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.