Chromatin Insulators for Human Gene Therapy

使用病毒载体的干细胞基因治疗具有两种类型的不必要的作用：由于周围异染色质的失活而导致载体沉默所引起的效力降低;或由载体附近的有害基因的载体增强子的激活所引起的遗传毒性。一般认为这些问题可以通过使用染色质绝缘体来避免。目前，对染色质绝缘子的了解有限，特别是哺乳动物和人类基因组的绝缘子。该项目的目标是，首先，使用我们团队开创的基因组方法来识别人类基因组中有可能充当染色质绝缘体的元素，其次，通过功能测定验证其绝缘体活性。具体而言：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，以确定两种蛋白质的所有结合位点， 在我们的数据库中包括的细胞系和原代细胞的基因组中，与染色质绝缘体CTCF和USF 1/2相关。2)假设染色质绝缘体本身作为DNA酶I超敏位点（DHS）放置在活性染色质结构域之间，我们将使用基因组高通量方法来识别潜在的组成性染色质绝缘体，这些基因间DHS存在于我们数据库的所有细胞系和主要细胞系的染色质谱中。我们还将通过分析原代人胎儿和成人红系细胞的DHS，并将位于活性红系基因结构域边界的红系细胞谱系特异性DHS确定为潜在的绝缘子，来检验谱系特异性染色质绝缘子的假设。然后，我们将信息比较的组成和 红系特异性DHS谱与CTCF和USF 1/2谱的比较，以鉴定显示所有三种特征的基因组元件和可能由染色质绝缘体显示的其他基因组特征，如特异性组蛋白修饰和基因组边界。3)我们将通过功能增强子阻断、染色质屏障和遗传毒性预防试验来验证所鉴定的元件的绝缘子功能。预计这些研究将确定用于基因治疗载体的新的有效绝缘子元件，并且还将提供关于人类基因组中染色质绝缘子的组织和功能的新信息。

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.