New Chromatin Insulators and Enhancers for Gene Therapy of the Hemoglobinopathies

摘要 该项目的目标是使用基因组方法在人类基因组中识别， 强大的染色质绝缘子和强大的红细胞特异性增强子。利用强大的 治疗性基因治疗载体中的绝缘体将通过降低 载体相关遗传毒性。强大的红细胞特异性增强剂的可用性将增加 基因治疗重型地中海贫血的疗效观察 通过对第一个特定目标的研究，我们将在细胞中发现强大的染色质绝缘体。 人类基因组使用新的基因组筛选方法，我们已经开发。特别 重点将放在确定强大的屏障绝缘体，也具有强大的 增强子阻断活性。这样的绝缘体被期望提高安全性和绝缘性。 整合基因治疗载体的功效。我们将确定的结构特征， 新的绝缘体，我们将研究其效力变化的结构基础。 在具体目标2中，我们将在人类中发现新的强大的红细胞特异性增强子。 利用分子和信息学方法对基因组进行分析。我们将验证红细胞谱系特异性 新的增强子，我们将表征它们，并将使用它们来识别分子 决定红细胞谱系特异性的特征。 通过对特异性目的3的研究，我们将产生新一代的珠蛋白基因治疗 向量。具体目标1中最强大的绝缘体将用于确定最佳的 大小、最高滴度和基因所需的最高遗传毒性保护 治疗载体特异性目标2或增强子盒的最强大的增强子将是 用于定量评估其支持珠蛋白基因表达的效力。的 最好的绝缘子和增强子将并入珠蛋白基因载体中， 将在地中海贫血患者的红系细胞中离体评估效力，并在体内评估效力 地中海贫血小鼠

ABSTRACT The goals of this project are to use genomic approaches to identify, in the human genome, powerful chromatin insulators and powerful erythroid specific enhancers. Use of powerful insulators in therapeutic gene therapy vectors will increase safety by decreasing the risks of vector-related genotoxicity. Availability of powerful erythroid specific enhancers will increase the efficacy of gene therapy in patients with severe thalassemia. With the studies of the first specific aim we will discover powerful chromatin insulators in the human genome using the novel genomic screening approaches we have developed. Special emphasis will be given in identifying powerful barrier insulators which also possess powerful enhancer blocking activity. Such insulators are expected to improve both the safety and the efficacy of integrating gene therapy vectors. We will determine the structural characteristics of the new insulators and we will investigate the structural basis of the variation in their potency. In specific aim 2 we will discover new powerful erythroid specific enhancers in the human genome using molecular and informatics methods. We will verify the erythroid lineage specificity of the new enhancers, we will characterize them and will use them to identify the molecular features that determine erythroid lineage specificity. With the studies of Specific Aim 3 we will produce a new generation of globin gene therapy vectors. The most powerful insulators of Specific aim 1 will be used to determine the optimal sizes, the highest titers and the highest protection from genotoxicity required for the gene therapy vectors. The most powerful enhancers of Specific Aim 2 or enhancer cassettes will be used for quantitative assessments of their potency in supporting globin gene expression. The best insulators and enhancers will be incorporated in globin gene vectors and their therapeutic potency will be assessed ex vivo in the erythroid cells of thalassemic patients and in vivo in thalassemic mice.