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（由申请人提供）：目前可用于修改表观基因组的工具是钝器，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造转基因动物或基因疗法，因此面临着转化为临床的艰难道路。因此，需要用于目标表观遗传操作的新技术，该技术应该具有基因特异性并且易于管理。考虑到这些品质，我们面临的挑战是开发一套基于 Piwi 相互作用 RNA (piRNA) 系统的工具，以准确诱导成体组织中目标位点的 DNA 甲基化，适用于所有哺乳动物。 PIWI 相互作用 RNA (piRNA) 代表了一种令人着迷的适应性机制，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 在海蛞蝓、小鼠和猕猴等动物的大脑和肾脏等多种体细胞组织中表达。激活成年哺乳动物的 piRNA 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。我们将使用此类 RNA 来开发针对特定基因和基因座的技术，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。虽然人们对生殖细胞中 piRNA 的整体沉默了解甚多，但对其在成体体组织中的活性知之甚少。我们的研究将验证它们作为技术平台或针对所有哺乳动物物种的任何基因的靶向表观遗传学的用途。模型生物的选择对于潜在表观遗传疗法的测试至关重要。在这些研究中，我们将使用 Agouti 活黄色小鼠，其毛色随着单个位点的 DNA 甲基化而变化。针对 Avy 转座子的诱导甲基化将为该位点的系统性分子沉默提供直接的视觉半定量证据。额外的实验将验证位点特异性和沉默程度。此外，我们将调整体细胞中的 piRNA 抑制系统以靶向基因区域和转座子。所产生的研究将提供急需的证据，阐明 piRNA 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 piRNA 靶向甲基化。