Chromatin Insulators for Human Gene Therapy

用于人类基因治疗的染色质绝缘体

• 批准号: 7799410
• 负责人: George Stamatoyannopoulos
• 金额: $ 48.82万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7799410
• 关键词: Adult; Animal Model; Binding; Binding Sites; Biological Assay; CCCTC-binding factor; Cell Line; Cell Lineage; Cells; Chromatin; Clinical; Data Set; Databases; Deoxyribonuclease I; Elements; Enhancers; Erythroid; Erythroid Cells; Exhibits; Gene Transduction Agent; Genes; Genome; Genomics; Goals; Heterochromatin; Histones; Human; Human Genome; Insulator Elements; Knowledge; Lentivirus Vector; Link; Mediating; Oncogene Activation; Patients; Positioning Attribute; Prevention; Proteins; Resolution; Site; Stem cells; Testing; Viral Vector; Zinc Fingers; base; chromatin immunoprecipitation; chromatin remodeling; fetal; gene therapy; genotoxicity; histone modification; in vivo; leukemia; promoter; therapeutic gene; tumor; vector

Stem cell gene therapy using viral vectors has two types of unwarranted effects: decreased efficacy caused by the silencing of the vectors due to inactivation by the surrounding heterochromatin; or genotoxicity caused by activafion by the vector's enhancers of deleterious genes in the vicinity of the vector. It is generally assumed that these problems can be avoided by the use of chromatin insulators. Currently there is limited knowledge about chromatin insulators in general and insulators of the mammalian and human genome in particular. The goal of this project is first, to use the genomic approaches our group has pioneered to identify elements of the human genome that have the potential to act as chromatin insulators and second, validate their insulator activity with functional assays. Specifically: 1) We will use a high resolution genomic chromatin immunoprecipitation approach. Chip sequencing, to identify all of the binding sites for two proteins known to be associated with chromatin insulators, CTCF and USF1/2, in the genomes of cell lines and primary cells included in our databases. 2) Under the hypothesis that chromatin insulators present themselves as DNAse I hypersensitive sites (DHSs) placed between active chromatin domains, we will use genomic high throughput approaches to identify as potential constitutive chromatin insulators those intergenic DHSs which are present in the chromafin profiles of all cell lines and primary cell lineages of our databases. We will also test the hypothesis of lineage-specific chromatin insulators by profiling the DHSs of primary human fetal and adult erythroid cells and identifying as potential insulators those erythoid lineage-specific DHSs which are located in the borders of active erythroid gene domains. We will then informafically compare the constitutive and erythroid-specific DHS profiles with the CTCF and USF1/2 profiles in order to identify genomic elements that exhibit all three features and other genomic features that may be exhibited by chromatin insulators, such as specific histone modifications and genomic boundaries. 3) We will validate the insulator funcfion of the so identified elements with functional enhancer-blocking, chromatin barrier and genotoxicity prevention assays. It is expected that these studies will identify new potent insulator elements for use in gene therapy vectors and will also provide new information about the organizafion and function of the chromatin insulators in the human genome.

使用病毒载体的干细胞基因治疗具有两种不必要的作用：由于周围异染色质失活而导致的载体导致载体导致的疗效降低；或由载体在载体附近的有害基因的增强子激活引起的遗传毒性。通常认为可以通过使用染色质绝缘子来避免这些问题。当前，有关哺乳动物和人类基因组绝缘剂的染色质绝缘子的知识有限。该项目的目的是首先使用我们的小组开创的基因组方法来确定人类基因组的元素，这些元素有可能充当染色质蛋白绝缘剂，其次，使用功能测定来验证其绝缘剂活性。具体：1）我们将使用高分辨率基因组染色质免疫沉淀方法。芯片测序，以识别两个已知是的蛋白质的所有结合位点 与我们的数据库中包含的细胞系和主要细胞基因组中，与染色质绝缘子CTCF和USF1/2相关。 2）假设染色质绝缘剂将其作为DNase I超敏位点（DHSS）放置在活性染色质结构域之间的假设，我们将使用基因组高通量方法来识别所有细胞系和我们数据蛋白酶的细胞线和原代细胞线酶中存在的基因间DHS的潜在染色质DHS。我们还将通过分析原代人胎儿和成年红细胞细胞的DHSS，并鉴定为潜在的绝缘剂，这些谱系特异性谱系特异性DHSS，这些DHSS位于活性红细胞基因域的边界中，我们还将检验谱系特异性染色质绝缘子的假设。然后，我们将与本规范性进行比较，并 带有CTCF和USF1/2谱的红细胞特异性DHS曲线，以识别具有染色质绝缘子（例如特定的组蛋白修饰和基因组边界）可能表现出的所有三个特征和其他基因组特征的基因组元素。 3）我们将验证具有功能增强子阻滞，染色质屏障和遗传毒性预防测定的SO鉴定元件的绝缘弹性。预计这些研究将确定用于基因治疗载体的新的有效绝缘元素，还将提供有关人基因组中染色质绝缘子的组织和功能的新信息。