GENE TRANSFER INTO HEMATOPOIETIC STEM CELLS

该项目致力于开发有效的策略，通过将治疗性基因转移到患有镰状细胞性贫血的小鼠和人类的干细胞中来纠正疾病表型。我们的目标是找到一种载体系统，能够在保持或扩大原始人类造血细胞数量和再繁殖潜力的条件下，实现相对有效的基因转移。第二个目标是开发一种治疗性表达盒，可用于直接增加转导细胞中的伽马mRNA的量，或通过表达转录因子、其跨显性突变体或针对相关mRNA的反义序列来激活内源性伽马-珠蛋白基因。第三个目标是在患有镰状细胞疾病的小鼠中建立基因治疗方案的模型，使用药物选择来扩增基因正常或基因纠正的少数造血细胞群体。在第一个特定目标下提出的研究旨在检验这样一个假设，即慢病毒载体固有的生物学优势，即前整合复合体的相对稳定性和这种核蛋白复合体横跨核膜的能力，将允许更高频率的原始人类移植进入再生细胞。还建议进行研究，以弥合我们对镰状细胞性贫血患者再生细胞的恢复和可转导性的认识上的差距。第二个具体目标是围绕逆转录病毒介导的基因转移到造血干细胞的基本假设而组织的，即可以使用逆转录病毒介导的基因转移在成熟的红细胞中达到治疗水平的伽马珠蛋白基因表达。两个战略将同时进行。1)开发产生高水平外源γ-珠蛋白mRNA的红系特异性表达盒，以及2)评估各种遗传元件激活外源γ-珠蛋白基因的能力。研究提出的特定目标3旨在使用人类血红蛋白疾病、严重的贝塔地中海贫血或镰状细胞疾病的小鼠模型，来测试这样一个假设，即通过逆转录病毒介导的基因转移经过基因修饰的少数细胞可以通过使用二氢叶酸还原酶选择系统进行药物选择来扩增，从而治愈这种血红蛋白疾病。建议进行实验，以在竞争性再繁殖的背景下大力测试选择系统，从而为未来为镰状细胞病患者制定基因治疗方案提供有用的信息。

This project is focused on the development of effective strategies for correcting the disease phenotype by therapeutic gene transfer into stem cells of mice and humans with sickle cell anemia. Our goals are to identify a vector system that is capable of achieving relatively efficient gene transfer into primitive human hematopoietic cells under conditions which preserve or expand their numbers and repopulating potential. A second goal is to develop a therapeutic expression cassette which can be used to directly increase the amount of gamma mRNA in transduced cells or to activate the endogenous gamma-globin genes through expression of a transcriptional factor, transdominant mutant therefor or antisense sequences targeted toward a relevant mRNA. A third goal is to model gene therapy protocols in mice with sickle cell disease using drug selection to amplify a genetically normal or gene corrected minority population of hematopoietic cells. The research proposed under the first specific aim is designed to test the hypothesis that the intrinsic biological advantages of lentiviral vectors, namely the relative stability of the pre-integration complex and the ability of this nucleoprotein complex do transverse the nuclear membrane, will allow a higher frequency of transduction of primitive human transfer into repopulating cells. Research is also proposed to close the gap in our knowledge regarding the recovery and transducibility of repopulating cells from patients with sickle cell anemia. The second specific aim is organized around the basic hypothesis that retroviral mediated gene transfer into hematopoietic stem cells can be used to achieve a therapeutic level of gamma-globin gene expression in maturing erythroblasts. Two strategies will be pursued concurrently. 1) development of an erythroid specific expression cassette that generates high levels of exogenous gamma-globin mRNA and 2) evaluation of various genetic elements with respect to their capacity to activate the exogenous gamma-globin genes. Research proposed specific aim 3 is designed to use murine models of human hemoglobin disorders, severe beta thalessemia or sickle cell disease, to test the hypothesis that a minority of cells that have been genetically modifier by retroviral mediated gene transfer can be amplified by drug selection using the dihydrofolate reductase selection system leading to cure of the hemoglobin disorder. Experiments are proposed to vigorously test the selection system in the context of competitive repopulation thereby providing useful information for developing gene therapy protocols for patients with sickle cell disease in the future.