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生细胞中，评估珠蛋白基因载体，开发激活内源性珠蛋白基因的策略，并评估珠蛋白基因治疗方法的安全性。这项拟议的研究涉及干细胞靶向基因转移到自体细胞，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在恒河猴模型之外使用培养的红系细胞、细胞系和小鼠模型进行。我们开发了一种基于猴免疫缺陷病毒(SIV)的慢病毒载体系统，它能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，获得约20%的转基因基因 血液学重建后的细胞。在具体目标1中，提出了实验的目标，目的是实现高水平的转导效率，从稳定状态的骨髓重新填充干细胞，同时将每个转导细胞的载体拷贝数限制在1个或尽可能少。还将努力高度浓缩干细胞，以便将输入的转导细胞限制在那些有可能有助于长期再繁殖的细胞。为了评估干细胞靶向基因转移的安全性，将对所有移植的动物进行长期监测，以了解含有载体基因组的造血细胞克隆的行为，该载体基因组已整合到原癌基因附近。在具体目标2中，实验旨在确定药物选择的造血干细胞是否会产生比未选择的干细胞表达更高水平的载体编码的伽马珠蛋白的红细胞。优化的珠蛋白载体 而旨在激活内源性伽马基因的载体将在培养的红系细胞中进行评估，以努力找到确保等于或&gt；20%HBF的载体设计。前景看好的治疗性珠蛋白载体将在恒河猴模型中进行体内测试。在特定的目标3中，我们将测试这样一种假设，即含有β-珠蛋白基因位点控制元件的载体将对细胞系和原代小鼠造血细胞中的周围基因表现出谱系受限的激活，这些基因可以被绝缘体阻断或减少。总体而言，我们的研究是 重点是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.