Y-Globin Gene Therapy Using In Vivo Selection

该项目的重点是开发一种使用y-珠蛋白的镰状细胞病基因治疗方法 慢病毒载体具有永久整合到造血干细胞（HSC）基因组中的能力。 从而提供了终身治愈的可能性。我们的努力将集中在满足两个关键的 这一做法最终取得成功的必要条件。首先是实现高水平的，持续的红细胞特异性 转移的7-珠蛋白表达盒的表达。最近，我们开发了一种7-珠蛋白 慢病毒载体具有在小鼠红细胞中实现10%水平的HbF的能力。因为很可能 镰状细胞病的治疗效果将需要每个细胞更高水平的HbF，第一个具体目标是 集中在修改我们的第一代载体，以进一步增加表达。这将通过一个 一系列精心设计的改变，旨在提高β-珠蛋白表达的水平和持久性。 经修饰以增强转录活性并抑制位置效应杂色和沉默的载体将 在体外和体内研究中进行评估。这些实验将最终测试治疗 在我们获得的两种镰状细胞病小鼠模型中，优化载体的功效。第二 具体目标集中于开发还含有选择性基因（甲基鸟嘌呤）的7-珠蛋白载体 甲基转移酶，MGMT），先前显示能够体内选择HSC。我们估计至少有10- 20%的能够产生表达T-珠蛋白的红细胞的HSC将需要用于治疗以下疾病： 镰状细胞病因此，在人体治疗试验中可能需要体内选择， 增加受体中亚治疗的、小比例的转导HSC，这将由有限的基因导致， 转移效率和非清髓性预处理的优选使用。体内选择实验 在具体目标2中提出了正常小鼠和两种镰状细胞海洋模型。最终目标是 以获得镰状细胞病模型中表达β-珠蛋白的细胞的治疗性体内选择。进展 这两个领域将对初始临床试验的规划产生重大影响， 在不久的将来，这种疗法更接近于成为镰状细胞病的潜在治疗方法。

This project is focused on the development of a gene therapy approach to sickle cell disease using a y-globin lentiviral vector with the capacity to permanently integrate into the _enome of hematopoietic stem cells (HSCs). thereby providing the possibility of a lifelong cure. Our efforts will concentrate on satisfying two critical requirements for the eventual success of this approach. The first is achieving high level, sustained erythroidspecific expression of a transferred 7-globin expression cassette. Recently, we have developed a 7-globin lentiviral vector with the capacity to achieve HbF at a level of 10% in the red cells of mice. Since it is likely that a therapeutic impact for sickle cell disease will require higher levels of HbF per cell, the first specific aim is centered on modifying our first generation vector to further increase expression. This will be done through a series of carefully planned alterations designed to boost both the level and persistence of ),-globin expression. Vectors modified to augment transcriptional activity and dampen position effect variegation and silencing will be evaluated in both in vitro and in viva studies. These experiments will culminate in testing the therapeutic efficacy of optimized vectors in two murine models of sickle cell disease which we have acquired. The second specific aim focuses on developing a 7-globin vector also containing a selectable gene (methylguanine methyltransferase, MGMT), previously shown to enable in viva selection of HSCs. We estimate that at least 10- 20% of HSCs capable of giving rise to T-globin expressing red cells will be required for a therapeutic effect in sickle cell disease. Therefore, it is likely that in viva selection will be needed in a human therapeutic trial to increase the subtherapeutic, small proportion of transduced HSCs in recipients that will result from limited gene transfer efficiency and the preferable use of non-myeloablative conditioning. In viva selection experiments in both normal mice and in the two sickle cell marine models are proposed in Specific Aim 2. The ultimate goal is to obtain therapeutic in viva selection of _,-globin expressing cells in the sickle cell disease models. Progress in these two areas would have substantial impact on the planning of initial clinical trials and would bring gene therapy closer to being a potential treatment for sickle cell disease in the near future.