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， 强大的染色质绝缘体和强大的红系特异性增强剂。使用强大的 治疗性基因治疗载体中的绝缘体将通过降低风险而增加安全性 媒介相关的遗传毒性。强大的红系特异性增强剂的可用性将增加 基因治疗对重型地中海贫血患者的疗效。 随着对第一个特定目标的研究，我们将发现在 使用我们开发的新的基因组筛选方法来筛选人类基因组。特价 重点是确定强大的阻挡绝缘体，这些绝缘体也具有强大的 增强阻断剂活性。这种绝缘子有望提高安全性和可靠性。 整合基因治疗载体的效果。我们将确定其结构特征 新的绝缘子，我们将调查其效力变化的结构基础。 在特定目标2中，我们将在人类体内发现新的强大的红系特异性增强剂。 利用分子和信息学方法研究基因组。我们将验证红系血统的特异性 在新的增强剂中，我们将对它们进行表征，并使用它们来鉴定分子 决定红系血统特异性的特征。 随着对特定目标3的研究，我们将产生新一代珠蛋白基因疗法。 向量。将使用特定目标1的最强大的绝缘体来确定最佳 大小、最高滴度和对基因所需的最高遗传毒性保护 治疗载体。特定目标2或增强剂盒中最强大的增强剂将是 用于定量评估它们支持珠蛋白基因表达的效力。这个 最好的绝缘体和增强剂将被整合到珠蛋白基因载体中，并用于治疗 将在体外评估地中海贫血患者的红系细胞的效力，并在体内评估 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.