Gene therapy of sickle cell disease through enhancement of fetal hemoglobin

这个项目的目标是开发方法，以获得治疗水平的嫁接，γ-珠蛋白 镰状细胞病患者中的载体慢病毒载体转导的造血干细胞（HSC） （SCD）。此外，我们将研究几种增加胎儿血红蛋白（HbF）积累的方法。 在正常的内源性水平下， 镰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 用能够在后代中高水平表达γ-珠蛋白的慢病毒载体转导的HSC 红系细胞，和2）开发多功能慢病毒载体以增强HbF表达。上 为了达到这个目的，我们将使用MGMT选择系统来选择γ-珠蛋白载体转导的HSC。 在上一个供资期内，这方面取得了重大进展，我们相信， 通过改进，将实现在大型动物模型中的HSC选择。此外，我们将调查 新的HOX融合蛋白NUP 98-HOXA 10是否可用于增加HSC基因转移， 扩增γ-珠蛋白载体转导的细胞用于移植。在第二个具体目标中， 提出了利用miRNA方法的实验来降低镰状β球蛋白的水平， 增强HbF的积累并提高治疗效果。我们还建议评估两个 永久性重新激活内源性γ-珠蛋白基因表达的能力的方法。 第一种方法将利用设计的锌指转录因子，其结合到转录因子中的-117位点。 γ-珠蛋白启动子。我们假设这将导致γ-珠蛋白基因的激活。第二 一种方法试图利用新鉴定的丙种球蛋白的mRNA介导的基因表达敲低 转录抑制因子BCL 1 IA，最近由StuartOrkin博士描述。通过这些努力，我们 寻求用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 在红细胞后代中有20%的内源性镰状血红蛋白（HbS）或更高。如果这些目标能够实现， 在SCD的人类基因治疗试验中取得成功似乎是可能的 相关性（见说明）： 我们的目标与公共卫生和国家心肺血液研究所的使命相关， 有效的干细胞靶向基因转移的发展将为许多遗传性血液病提供治疗。 疾病由于镰状细胞病会导致严重的症状，残疾和早期死亡， 迫切需要诸如基因疗法的疗法。

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.