Gene therapy of sickle cell disease through enhancement of fetal hemoglobin

这个项目的目标是开发方法来获得植入的治疗水平的丙种球蛋白。 载体慢病毒载体转导的造血干细胞在镰状细胞病患者中的应用 (SCD)。此外，我们还将研究几种增加胎儿血红蛋白(HBF)蓄积的方法。 在正常的内源性水平的背景下，由于伽玛珠蛋白转基因表达而产生的 镰刀状珠蛋白链。我们的具体目标是：1)获得治疗相关水平的造血干细胞 转导慢病毒载体的细胞(HSCs)在子代中高水平表达γ-珠蛋白 2)开发多功能慢病毒载体以增强HBF的表达。在第一个 目的：我们将使用MGMT选择系统来选择伽马珠蛋白载体转导的造血干细胞。 在上一个供资期间，在这一领域取得了实质性进展，我们认为， 改进后，HSC在大动物模型中的选择将实现。此外，我们还将调查 一种新的HOX融合蛋白NUP98-HOXA10是否可以用于增加HSC的基因转移和 用于移植的伽玛珠蛋白载体转导细胞的扩增。在第二个具体目标中， 利用miRNA方法的实验被提出以降低镰状贝塔珠蛋白的水平，从而 增强HbF蓄积，增强疗效。我们还建议评估两个 永久重新激活内源性伽马珠蛋白基因表达的能力的方法。 第一种方法将利用一种设计的锌指转录因子，它结合到 丙种球蛋白促进剂。我们假设这将导致伽马珠蛋白基因的激活。第二 方法寻求利用mlRNA介导的基因表达敲除新发现的丙种球蛋白 转录抑制因子BCL1IA，最近由Stuart Orkin博士描述。通过这些努力，我们 寻求通过珠蛋白载体修饰的细胞和HBF表达获得至少20%的HSC植入水平 在红细胞子代中的内源性镰状血红蛋白(HBS)占20%或更高。如果这些目标能够实现， SCD的人类基因治疗试验似乎有可能取得成功 相关性(请参阅说明)： 我们的目标与公共卫生和国家心肺血液研究所的使命有关，因为 有效的干细胞靶向基因转移的发展将为许多遗传血液提供治疗 疾病。因为镰状细胞病会导致严重的症状、残疾，而且往往会过早死亡，治愈 迫切需要基因疗法等疗法。

The goals of this project are to develop methods to obtain therapeutic levels of engrafted, gamma-globin vector lentiviral vector-transduced hematopoietic stem cells (HSCs) in patients with sickle cell disease (SCD). Additionally, we w/ill study several approaches to augment accumulation of fetal hemoglobin (HbF) resulting from gamma-globin transgene expression in the context ofthe normal endogenous levels ofthe sickle globin chain. Our specific aims are: 1) to obtain therapeutically relevant levels of hematopoietic stem cells (HSCs) transduced with a lentiviral vector capable of high level, gamma-globin expression in progeny erythroid cells, and 2) to develop multifunctional lentiviral vectors to enhance HbF expression. In the first aim, we will use the MGMT selection system to enable selection of gamma-globin vector-transduced HSCs. Substantial progress was made in this area in the last funding period and we believe, with further improvements, HSC selection in a large animal model will be achieved. Additionally, we will investigate whether a novel HOX fusion protein, NUP98-HOXA10, can be used to increase HSC gene transfer and expansion of gamma-globin vector transduced cells for transplantation. In the second specific aim, experiments utilizing an miRNA approach are proposed to reduce the levels of sickle beta globin so to enhance the accumulation of HbF and augment therapeutic efficacy. We also propose to evaluate two approaches for the ability to permanently re-activate expression of the endogenous gamma-globin genes. The first approach will utilize a designer zinc-finger transcription factor which binds to the -117 site in the gamma-globin promoter. We hypothesize this will lead to activation ofthe gamma-globin gene. The second approach seeks to utilize mlRNA-mediated gene expression knockdown of the newly identified gammaglobin transcriptional repressor BCL1 IA, recently described by Dr. Stuart Orkin. Through these efforts, we seek to obtain HSC engraftment levels of at least 20% with globin-vector modified cells and HbF expression in the red cell progeny of 20% of endogenous sickle hemoglobin (HbS) or higher. If these goals can be met, success in a human gene therapy trial for SCD would seem likely RELEVANCE (See instmctions): Our goals are relevant to public health and the mission of the National Heart, Lung and Blood Institute since the development of effective stem cell targeted gene transfer would provide therapy for many inherited blood diseases. Because sickle cell disease causes severe symptoms, disability and often early death, curative therapies such as gene therapy are urgently needed.