Gene Transfer into Hematopoietic Stem Cells

基因转移至造血干细胞

• 批准号: 6967748
• 负责人: ARTHUR W. NIENHUIS
• 金额: $ 29.47万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6967748
• 关键词: Lentivirus; Macaca mulatta; biotechnology; cell population study; disease /disorder model; erythropoiesis; gene expression; gene therapy; genetic regulatory element; globin; hematopoietic stem cells; hemoglobin F; human subject; laboratory mouse; messenger RNA; patient oriented research; polymerase chain reaction; recombinant virus; sickle cell anemia; sickling inhibitor; simian immunodeficiency virus; therapy design /development; tissue /cell culture; transfection; transfection /expression vector

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.

该项目的重点是优化慢病毒载体介导的基因转移到长期重脑细胞中，评估环球蛋白基因载体，用于制定激活内源性球蛋白基因并评估环球蛋白基因疗法方法的策略。提出的研究涉及干细胞靶向基因转移到自体细胞中，这些基因将在恒河体移植模型中进行，而除了恒河类模型之外，还将使用培养的红细胞细胞，细胞系和鼠模型来进行优化球蛋白载体并评估其安全性的策略。我们已经建立了一种基于猿猴免疫缺陷病毒（SIV）的慢病毒载体系统，该病毒有效地将基因转移到动员的细胞因子中，动员的恒河猴的外周血干细胞产生了大约20％的遗传改性 血液学重建后的细胞。在特定的目标1中，提出了实验，其目标是将高水平的转导效率从稳态骨髓骨髓中重新填充，同时将矢量拷贝数限制为1或每次转导的细胞尽可能少。还将努力以高度富集干细胞，以使输注的转导细胞仅限于有可能有助于长期重生的细胞。为了评估靶向干细胞靶向基因转移的安全性，将对所有移植动物的行为进行长期监测，以对含有载体基因组的造血细胞的克隆进行行为，该载体基因组已在原型癌中综合。在特定的目标2中，设计实验旨在确定药物选择的造血干细胞是否会产生与非选择的干细胞相比，表达更高水平的载体编码γ-球蛋白的红细胞。优化的球蛋白向量 旨在激活内源性伽马基因的载体将在培养的红细胞细胞中进行评估，以找到确保等于或> 20％HBF的矢量设计。有希望的治疗球蛋白载体将在恒河类模型中在体内进行测试。在特定的目标3中，我们将测试以下假设：含有β-珠蛋白基因座控制元件的载体将在细胞系中表现出周围基因的谱系限制激活，而原代鼠造血细胞可以通过绝缘子阻止或减少。总体而言，我们的研究是 专注于开发有效的镰状细胞疾病基因治疗方法，同时评估这些方法的安全性。