Development of piRNAs for target-specific methylation

 描述（由申请人提供）：目前可用的修饰表观基因组的工具是钝的工具，而不是研究人员和临床医生都非常需要的精确编辑器。现有的靶向方法依赖于创建转基因动物或基因治疗，因此面临着转化为临床的艰难道路。因此，需要新的靶表观遗传操作技术，该技术应该是基因特异性的并且易于管理。考虑到这些品质，我们的挑战是开发一套基于Piwi相互作用RNA（皮尔纳）系统的工具，以准确诱导成人组织中靶位点的DNA甲基化，适用于所有哺乳动物。PIWI相互作用RNA（piRNA）代表了一种迷人的适应机制，也是基因特异性抑制创新的潜在“现成”工具。最初，piRNA及其相关蛋白被认为仅在生殖细胞中表达，然而，越来越多的证据发现piRNA在从海蛞蝓到小鼠到猕猴的动物的体细胞组织中的表达与脑和肾一样多样。在成年哺乳动物中激活皮尔纳通路有望缩小基础研究与人类应用之间的差距。与广泛使用的miRNA/siRNA治疗相比，piRNA沉默的关键区别在于piRNA提供敏感的序列特异性并诱导DNA甲基化。我们将使用这类RNA来开发靶向特定基因和位点的技术，以在预定的基因组位置实现稳定的、有丝分裂可遗传的沉默。虽然对生殖细胞中piRNA的整体沉默了解很多，但对它们在成年体细胞组织中的活性了解甚少。我们的研究将验证它们作为技术平台或靶向表观遗传学在所有哺乳动物物种的任何基因中的用途。模式生物的选择在潜在的表观遗传疗法的测试中是至关重要的。在这些研究中，我们将使用Agglutinase可存活的黄色小鼠，其毛色变化伴随着单个基因座的DNA甲基化。靶向Avy转座子的诱导甲基化将提供在该位点的系统性分子沉默的直接视觉半定量证据。其他实验将验证位点特异性和沉默程度。此外，我们将调整索马中的皮尔纳抑制系统以靶向基因区域以及转座子。所产生的研究将提供急需的证据，阐明皮尔纳在哺乳动物体细胞组织中的作用和活性，并将用于为更广泛的研究和治疗社区开发皮尔纳靶向甲基化。

 DESCRIPTION (provided by applicant): Currently available tools to modify the epigenome are blunt instruments rather than the precise editors in great demand for both researchers and clinicians. Existing targeted approaches rely on creating transgenic animals or gene therapy, and therefore face a difficult path for translation to the clinic. Thus, new technology for targete epigenetic manipulation is needed that should be both gene-specific and easy to administer. With these qualities in mind, our challenge is to develop a suite of tools, based on the Piwi-interacting RNA (piRNA) system, to accurately induce DNA methylation of targeted loci in adult tissues, applicable to all mammals. PIWI-interacting RNAs (piRNAs) represent a fascinating adaptive mechanism and a potentially "ready made" tool for innovation in gene-specific repression. Originally piRNAs and their associated proteins were thought to be expressed only in germ cells, however, mounting evidence finds the expression of piRNAs in somatic tissues as diverse as brain and kidney in animals from sea slug to mouse to macaque. Activating the piRNA pathway in adult mammals holds promise in closing the gap between basic research and human application. The crucial difference in silencing by piRNAs compared to widely used miRNA/siRNA treatment is that piRNAs offer sensitive sequence specificity and induce DNA methylation. We will use this class of RNA to develop the technology to target specific genes and loci for stable, mitotically heritable, silencing at pre-determined genomic locations. While much is known about global silencing by piRNAs in germ cells, much less is known about their activity in adult somatic tissues. Our studies will validate their use as a technological platform or targeted epigenetics in any gene for all mammalian species. Choice of model organism is critical in the testing of potential epigenetic therapeutics. For these studies, we will use the Agouti viable yellow mouse, which varies in coat color concomitantly with DNA methylation at a single locus. Induced methylation targeted to the Avy transposon will provide direct visual semi-quantitative evidence of systemic molecular silencing at this locus. Additional experiments will verify the site specificity and degree of silencing. Further, we will adapt the piRNA suppression system in the soma to target genic regions as well as transposons. The research generated will provide sorely needed evidence clarifying the roles and activity of piRNA in somatic tissues of mammals and will be used to develop piRNA targeted methylation for the wider research and therapeutic communities.