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（由适用提供）：当前修改表观基因组的工具是钝器，而不是对研究人员和临床医生的巨大需求的精确编辑。现有的靶向方法依赖于创建转基因动物或基因疗法，因此面临着转化为诊所的困难途径。这是需要针对靶向表观遗传操作的新技术，应该既应该是基因特异性且易于管理的。考虑到这些品质，我们的挑战是根据PIWI相互作用RNA（PIRNA）系统开发一套工具，以准确诱导成人组织中靶向位置的DNA甲基化，适用于所有哺乳动物。 PIWI相互作用的RNA（PIRNA）代表了一种引人入胜的适应性机制，也是一种潜在的“现成”工具，用于基因特异性表达中的创新。最初认为PIRNA及其相关蛋白仅在生殖细胞中表达，但是，越来越多的证据发现，从海sl到小鼠再到猕猴的动物中的脑和肾脏，如大脑和肾脏中的piRNA表达。激活成年哺乳动物的PIRNA途径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处理相比，PIRNA沉默的关键差异是PIRNA提供了敏感的序列特异性并诱导DNA甲基化。我们将使用这类RNA来开发该技术，以靶向特定的基因和位置，以在预定的基因组位置稳定，有线遗传，沉默。尽管对生殖细胞中的PIRNA的全球沉默已知，但对它们在成年体细胞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的使用或所有哺乳动物物种中任何基因的靶向表观遗传学。模型生物的选择对于测试潜在表观遗传疗法至关重要。在这些研究中，我们将使用Agouti可行的黄色小鼠，它们的外套颜色与单个基因座的DNA甲基化伴随着不同。针对Avy转座子的诱导甲基化将提供直接的视觉半定量证据，证明该基因座的全身分子沉默。其他实验将验证现场特异性和沉默程度。此外，我们将适应躯体中的PIRNA抑制系统，以靶向遗传区域和转座子。所产生的研究将提供急需的证据，以阐明piRNA在哺乳动物体细胞组织中的作用和活性，并将用于开发针对更广泛的研究和治疗群落的PIRNA靶向甲基化。