GENE THERAPY FOR SICKLE CELL DISEASE

本项目的研究围绕我们的假设进行，即镰状细胞病的生理基因治疗可以通过基因逆转从胎儿（HbF）到成人（HbS）血红蛋白的转换来实现，通过基因转移到造血干细胞。在未来五年的研究中，我们的目标是通过对控制红细胞形成的分子机制、决定γ和β-珠蛋白表达比例的调节因子以及调节干细胞行为和转导的生物学控制的理解，显著推进基因治疗的发展。“红细胞生成中Epo受体的信号传导，拟议的研究旨在通过定义Jak-2远端的信号传导组分来理解从促红细胞生成素受体发出的途径，Jak-2与受体的膜近端部分相互作用，并定义受体膜远端部分的冗余和非冗余功能。“红细胞生成中的造血环指1（HERF1）”，研究重点是定义HERF1的作用，这是一种新型的红细胞特异性RING蛋白，从而深入了解控制成红细胞成熟终末阶段的分子途径。"Identification and Characterization of Factors which Modulate gamma-Globin Gene Expression"，实验集中于表征与转换机制有关的蛋白质，并确定这些蛋白质是否可用于调节成熟成红细胞中γ和β合成的相对平衡。已经开发了一种基于二氢叶酸还原酶变体的选择系统"转导的造血干细胞的体内选择"，其允许扩增遗传修饰的造血细胞。现在将在鼠模型中研究这种扩增的机制，并且DHFR选择系统也将在最终尝试将这种方法推进到临床应用的背景下适用于非人灵长类动物模型。 “基因转移到造血干细胞”，提出的实验集中在标准（MuLV）逆转录病毒载体慢病毒载体的相对能力，以转移基因到原始的再生细胞从镰状细胞病患者和成熟的成红细胞表达治疗基因的评价。对人类血红蛋白疾病动物模型的研究将检验逆转录病毒介导的基因转移和基因修饰细胞群体的扩增可用于有效纠正疾病表型的假设。该研究得到了一个行政核心和三个科学核心的支持，提供纯化的干细胞，标准化的载体制剂或获得独特的动物模型。通过这一协调的研究计划，我们预计将朝着镰状细胞病基因治疗成功的最终目标取得实质性进展。

The research encompassed within this program project is organized around our hypothesis that physiol9ogical gene therapy for sickle cell disease may be achieved by genetically reversing the switch from fetal (HbF) to adult (HbS) hemoglobin through gene transfer into hematopoietic stem cells. During the next five years of research, our goal is to significantly advance the de4velopment of gene therapy by achieving a growing understanding of the molecular mechanisms that control red cell formation, the regulatory factors that determine the proportion of gamma and beta-globin expression and the biological controls that modulate stem cell behaviors and transduction. "Signaling by the Epo Receptor in Erythropoiesis, the proposed research seeks to understand the pathways that emanate from the erythropoietin receptor by defining signaling components distal to Jak-2, which interacts with the membrane proximal portion of the receptor, and to define both redundant and non-redundant functions of the membrane distal part of the receptor. "Hematopoietic RING Finger 1 (HERF1) in Erythropoiesis", the research is focused around defining the role of HERF1 a novel, erythroid specific RING protein, thereby gaining insights into the molecular pathways that control the terminal stages of erythroblast maturation. "Identification and Characterization of Factors which Modulate gamma-Globin Gene Expression", experiments are focused on characterizing proteins that have been implicated in switching mechanisms and determining whether such proteins can be used to modulate the relative balance of gamma and beta synthesis in maturing erythroblasts. "In Vivo Selection of Transduced Hematopoietic Stem Cells", a selection system based on variants of dihydrofolate reductase, has been developed that allows amplification of genetically modified hematopoietic cells. The mechanism of such amplification will now be investigated in the murine model and the DHFR selection system will also be adapted for use in a non-human primate model in the context of ultimately attempting to advance this approach to clinical use. "Gene Transfer into Hematopoietic Stem Cells," the proposed experiments are focused on evaluation of standard (MuLV) retroviral vectors to lentiviral vectors with respect to their relative ability to transfer genes into primitive repopulating cells from patients with sickle cell disease and to express a therapeutic gene in maturing erythroblasts. Research in animal models of human hemoglobin disorders will test the hypothesis that retroviral mediated gene transfer and amplification of a population of genetically modified cells can be used to effectively correct the disease phenotype. The research is supported by an Administrative Core and three scientific Core's that provide purified stem cells, standardized vector preparations or access to unique animal models. Through this coordinated program of research we anticipate substantial progress toward the ultimate goal of successful gene therapy for sickle cell disease.