Gene Transfer into Hematopoietic Stem Cells

该项目的重点是优化慢病毒载体介导的基因转移到长期再增殖细胞，珠蛋白基因载体的评估，开发策略，以激活内源性珠蛋白基因和评估珠蛋白基因治疗方法的安全性。拟议的研究涉及干细胞靶向基因转移到自体细胞中，这将在恒河猴移植模型中完成，而优化珠蛋白载体并评估其安全性的策略将使用培养的红系细胞，细胞系和小鼠模型以及恒河猴模型进行。我们已经开发了一种基于猴免疫缺陷病毒（SIV）的慢病毒载体系统，该系统有效地将基因转移到细胞因子动员的恒河猴外周血干细胞中， 血液重建后的细胞。在具体目标1中，提出了实验的目标是实现高水平的转导效率，从稳态骨髓中重新增殖干细胞，同时将载体拷贝数限制为1个或尽可能少的每个转导细胞。还将努力高度富集干细胞，使得输注的转导细胞可以限于具有有助于长期再增殖的潜力的那些。为了评价干细胞靶向基因转移的安全性，将长期监测所有移植动物的造血细胞克隆的行为，所述造血细胞克隆含有在原癌基因附近整合的载体基因组。在具体目标2中，设计实验以确定药物选择的造血干细胞是否将产生表达比非选择的干细胞更高水平的载体编码的γ-珠蛋白的成红细胞。优化的珠蛋白载体 并且在培养的红系细胞中评价设计用于激活内源性γ基因的载体，以努力找到确保等于或> 20%HbF的载体设计。有前途的治疗珠蛋白载体将在恒河猴模型中进行体内测试。在具体目标3中，我们将检验以下假设：含有β-珠蛋白基因座控制元件的载体将表现出细胞系和原代鼠造血细胞中周围基因的谱系限制性激活，其可以被绝缘子阻断或减弱。总的来说，我们的研究 致力于开发镰状细胞病基因治疗的有效方法，同时评估这些方法的安全性。

This project is focused on optimizing lentiviral vector mediated gene transfer into long-term repopulating cells, on the evaluation of globin gene vectors, on developing strategies to activate the endogenous globin genes and on evaluating the safety of globin gene therapy approaches. The proposed research involves stem cell targeted gene transfer into autologous cells which will be done in a rhesus transplantation model whereas strategies to optimize globin vectors and to evaluate their safety will be performed using cultured erythroid cells, cell lines and murine models in addition to the rhesus model. We have developed a lentiviral vector system based on simian immunodeficiency virus (SIV) that efficiently transfers genes into cytokine mobilized, peripheral blood stem cells of rhesus macaques yielding approximately 20% genetically modified cells following hematologic reconstitution. In Specific Aim 1, experiments are proposed with a goal of achieving a high level of transduction efficiency into repopulating stem cells from steady state bone marrow while limiting vector copy number to 1 or as few as possible per transduced cell. Efforts will be also be made to highly enrich stem cells so that the transduced cells infused can be limited to those which have the potential to contribute to long-term repopulation. To evaluate the safety of stem cell-targeted gene transfer, all transplanted animals will be monitored long-term for the behavior of clones of hematopoietic cells containing a vector genome which has integrated near a proto-oncogene. In Specific Aim 2, experiments are designed to determine whether drug selected hematopoietic stem cells will generate erythroblasts expressing higher levels of vector encoded gamma-globin than non-selected stem cells. Optimized globin vectors and vectors designed to activate the endogenous gamma genes will be evaluated in cultured erythroid cells in an effort to find a vector design which ensures equal to or >20% HbF. Promising therapeutic globin vectors will be tested in vivo in the rhesus model. In Specific Aim 3, we will test the hypothesis that the vectors containing beta-globin locus control elements will exhibit lineage-restricted activation of surrounding genes in cell lines and primary murine hematopoietic cells that can be blocked or diminished by an insulator. Overall, our research is focused on developing effective approaches for gene therapy of sickle cell disease while simultaneously evaluating the safety of these approaches.