Selectable Gamma-Globin Lentiviral Vectors for SCD

描述(申请人提供)：该项目专注于开发一种使用伽马珠蛋白慢病毒载体的镰状细胞疾病的基因治疗方法，该载体能够永久整合到造血干细胞(HSCs)的基因组中，从而提供终身治愈的可能性。我们的努力将集中于满足这一办法最终取得成功的两个关键要求。第一个是获得高水平的、持续的红系特异性表达转移的伽马珠蛋白表达盒。最近，我们开发了一种伽玛珠蛋白慢病毒载体，能够在小鼠的红细胞中达到10%的HBF水平。由于镰状细胞病的治疗效果可能需要每个细胞更高水平的HBF，因此第一个具体目标是修改我们的第一代载体以进一步提高表达。这将通过一系列精心计划的改变来实现，旨在提高伽马珠蛋白的表达水平和持久性。将在体外和体内研究中对载体进行修改，以增强转录活性和抑制位置效应、杂化和沉默。这些实验最终将在我们已经获得的两种镰状细胞病小鼠模型中测试优化载体的治疗效果。第二个具体目标是开发一种也包含可选择基因(甲基鸟嘌呤甲基转移酶，MGMT)的伽玛珠蛋白载体，此前已证明该基因能够在体内选择造血干细胞。我们估计，至少10%-20%的能够产生表达丙种球蛋白的红细胞的造血干细胞将需要对镰状细胞疾病的治疗效果。因此，很可能需要在人体治疗试验中进行体内选择，以增加受者亚治疗性、小比例转导的HSCs，这是由于有限的基因转移效率和非清髓性调节的优选使用所致。在正常小鼠和两种镰状细胞病小鼠模型中的体内选择实验是有针对性的2.最终目的是在镰状细胞病模型中获得体内治疗性的γ-珠蛋白表达细胞的选择。这两个领域的进展将对初步临床试验的规划产生重大影响，并将使基因疗法更接近于在不久的将来成为治疗镰状细胞疾病的潜在疗法。

DESCRIPTION (provided by applicant): This project is focused on the development of a gene therapy approach to sickle cell disease using a gamma-globin lentiviral vector with the capacity to permanently integrate into the genome 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 erythroid-specific expression of a transferred gamma-globin expression cassette. Recently, we have developed a gamma-globin lentiviral vector with the capacity to achieve HbF level of 10 percent 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 gamma-globin expression. Vectors modified to augment transcriptional activity and dampen position effect variegation and silencing will be evaluated in both in vitro and in vivo 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 gamma-globin vector also containing a selectable gene (methylguanine methyltransferase, MGMT), previously shown to enable in vivo selection of HSCs. We estimate that at least 10-20 percent of HSCs capable of giving rise to gamma-globin expressing red cells will be required for a therapeutic effect in sickle cell disease. Therefore, it is likely that in vivo 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 vivo selection experiments in both normal mice and in the two sickle cell murine models are proposed in Specific Aim 2. The ultimate goal is to obtain therapeutic in vivo selection of y-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.