GENE TRANSFER INTO HEMATOPOIETIC STEM CELLS

该项目的重点是通过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的有效策略的开发。我们的目标是确定一个载体系统，能够实现相对有效的基因转移到原始的人类造血细胞的条件下，保持或扩大其数量和重新填充的潜力。第二个目标是开发一种治疗性表达盒，其可用于直接增加转导细胞中γ mRNA的量或通过表达转录因子、其反式显性突变体或靶向相关mRNA的反义序列来激活内源性γ-珠蛋白基因。第三个目标是在患有镰状细胞病的小鼠中模拟基因治疗方案，使用药物选择来扩增造血细胞的遗传正常或基因校正的少数群体。在第一个具体目标下提出的研究旨在检验慢病毒载体的内在生物学优势（即整合前复合物的相对稳定性和该核蛋白复合物确实穿过核膜的能力）将允许将原始人转移更高频率地转导到重建细胞中的假设。研究还建议关闭差距，在我们的知识有关的恢复和转导的再生细胞从镰状细胞性贫血患者。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中可用于在成熟成红细胞中实现治疗水平的γ-珠蛋白基因表达的基本假设组织的。将同时采取两项战略。1)产生高水平外源γ-珠蛋白mRNA的红细胞特异性表达盒的开发和2）评价各种遗传元件激活外源γ-珠蛋白基因的能力。研究提出的具体目标3旨在使用人类血红蛋白疾病、重度β地中海贫血或镰状细胞病的小鼠模型，以检验以下假设：通过逆转录病毒介导的基因转移进行遗传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而扩增，从而治愈血红蛋白疾病。实验中提出了大力测试的选择系统的竞争性再增殖的背景下，从而提供有用的信息，为镰状细胞病患者的基因治疗方案在未来的发展。

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.