Development of piRNAs for target-specific methylation

开发用于靶标特异性甲基化的 piRNA

• 批准号: 8947514
• 负责人: Dana Dolinoy
• 金额: $ 58.84万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-30 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8947514
• 关键词: Acute; Adult; Affect; Age; Aging; Alcohol consumption; Animal Model; Animals; Basic Science; Belief; Biological Assay; Biological Models; Biosensor; Brain; Brain-Derived Neurotrophic Factor; Chronic; Clinic; Clinical; Clinical Sciences; Color; DNA; DNA Methylation; Development; Disease; Effectiveness; Epigenetic Process; Equilibrium; Exhibits; Face; Gene Expression; Gene Silencing; Gene Targeting; Genes; Genetic Transcription; Genome; Genomic Instability; Genomics; Germ Cells; Germ Lines; Human; Hypermethylation; Injection of therapeutic agent; Kidney; Laboratories; Location; Macaca; Maintenance; Malignant Neoplasms; Mammals; Measures; Methods; Methylation; MicroRNAs; Mind; Modification; Molecular; Mus; Pathology; Pathway interactions; Pharmacologic Substance; Phenotype; Positioning Attribute; Proteins; RNA; Radiation; Repression; Research; Research Personnel; Rett Syndrome; Role; Site; Small Interfering RNA; Small RNA; Specificity; System; Technology; Testing; Therapeutic; Therapeutic community technique; Time; Tissues; Transcript; Transgenic Animals; Translations; Visual; Work; base; biological adaptation to stress; clinical application; congenic; demethylation; environment related cancer; epigenome; epigenomics; experience; fascinate; gene therapy; human disease; innovation; instrument; mouse model; neuronal cell body; new technology; novel; nutrition; public health relevance; research study; sea slug; tool; toxicant

 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.

 描述(申请人提供)：目前可用来修改表观基因组的工具是钝化的仪器，而不是对研究人员和临床医生都有很大需求的精确编辑。现有的靶向方法依赖于创造转基因动物或基因治疗，因此面临着转化为临床的困难路径。因此，靶向表观遗传学操作的新技术需要既具有基因特异性又易于管理。考虑到这些特点，我们的挑战是开发一套基于Piwi相互作用RNA(PiRNA)系统的工具，以准确地诱导成年组织中目标基因的DNA甲基化，适用于所有哺乳动物。Piwi相互作用RNAs(PiRNAs)代表了一种迷人的适应机制，也是一种潜在的“现成”工具，用于基因特异性抑制的创新。最初，piRNAs及其相关蛋白被认为只在生殖细胞中表达，然而，越来越多的证据发现，piRNAs在动物的大脑和肾脏等身体组织中的表达多种多样，从海懒到小鼠再到猕猴。在成年哺乳动物中激活piRNA途径有望缩小基础研究和人类应用之间的差距。与广泛使用的miRNA/siRNA治疗相比，piRNAs沉默的关键区别在于，piRNAs提供了敏感的序列特异性，并诱导了DNA甲基化。我们将使用这类RNA来开发针对特定基因和位点的技术，以在预定的基因组位置稳定地、有丝分裂地遗传、沉默。虽然人们对生殖细胞中piRNAs的整体沉默知之甚多，但对其在成年体细胞组织中的活动却知之甚少。我们的研究将验证它们在所有哺乳动物物种的任何基因中作为技术平台或靶向表观遗传学的用途。模式生物的选择在潜在的表观遗传疗法的测试中至关重要。在这些研究中，我们将使用Agti存活的黄色小鼠，它的毛色变化伴随着单个基因座上的DNA甲基化。针对Avy转座子的诱导甲基化将提供系统分子沉默在该基因座的直接视觉半定量证据。额外的实验将验证位置的特异性和沉默的程度。此外，我们将使SOMA中的piRNA抑制系统针对基因区域和转座子。这项研究将提供迫切需要的证据，阐明piRNA在哺乳动物躯体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piRNA靶向甲基化。