FLT LIGAND PERIPHERAL BLOOD STEM CELL MOBILIZATION FOR STEM CELL GENE THERAPY

用于干细胞基因治疗的 FLT 配体外周血干细胞动员

• 批准号: 6116549
• 负责人: M ROSENZWEIG
• 金额: $ 12.84万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6116549
• 关键词: Mammalia; animal tissue; gene therapy; hematology; immunology; inflammation

Optimization of the harvesting and transduction of hematopoietic stem cells is critical to successful stem cell gene therapy We evaluated the utility of a novel protocol involving flt-3 and G-CSF mobilization of peripheral blood stem cells and retroviral transduction using hematopoietic growth factors to introduce a reporter gene, murine CD24, into hematopoietic stem cells in nonhuman primates Rhesus macaques were treated with flt-3 ligand (200 mg/kg) and G-CSF (20 mg/kg) for 7 days and autologous CD34+ peripheral blood stem cells harvested by leukapheresis CD34+ cells were transduced with an MFG-based retroviral vector encoding murine CD24 using 4 daily transductions with centrifugations in the presence of flt-3 ligand (100 ng/ml), SCF (100 ng/ml) and PIXY (100 ng/ml) in serum free medium Animals were sublethally irradiated (400 cGy) on the day of transplantation Engraftment was monitored sequentially in the bone marrow and blood using both multiparameter flo w cytometry and semi-quantitative DNA PCR Our data demonstrate successful and persistent engraftment in multiple hematopoietic lineages, including granulocytes, monocytes and B and T lymphocytes At 46 and 117 days post transplantation 75% and 6% of granulocytes, respectively, expressed mCD24 antigen at the cell surface Peak in vivo levels of genetically-modified peripheral blood lymphocytes (PBL) approached 60% as assessed both by flow cytometry and PCR 18 days post transplantation In addition, naive (CD45RA+ and CD62L+) CD4+ and CD3+8+ cells were the predominant phenotype of the T cells detected at this time Engraftment has subsequently plateaued but persisted at between 10 and 15% of PBLs for 133 days Acytotoxic T cell response against murine CD24 was detected in only one animal This degree of persistent long-lived, high level gene marking of multiple hematopoietic lineages, including naive T cells, has important implications for the field of stem cell gene therapy

造血细胞采集和转导条件的优化 干细胞是干细胞基因治疗成功的关键 评价一种涉及Flt-3和G-CSF的新方案的实用性 外周血干细胞和逆转录病毒的动员 使用造血生长因子的转导作用 报告基因小鼠CD24在非人类体内转化为造血干细胞 用Flt-3配体(200 mg/kg)处理灵长类猕猴 G-CSF(20 mg/kg)，连续7天和自体CD34+外周血 用白细胞分离法分离CD34+细胞获得的干细胞 编码小鼠CD24的MFG逆转录病毒载体 Flt-3配基存在下的离心法转导 (100 ng/ml)、SCF(100 ng/ml)和Pixy(100 ng/ml)。 动物受到亚致死剂量的辐射(400cGy.) 在骨骼中连续监测移植植入率 用多参数流式细胞术检测骨髓和血液 我们的数据证明了半定量DNA聚合酶链式反应的成功和 在多个造血系中持续植入，包括 46日龄和117日龄粒细胞、单核细胞和B、T淋巴细胞 移植后分别为75%和6%的粒细胞， 在体内细胞表面表达mCD24抗原的高峰水平 转基因外周血淋巴细胞(PBL)接近60% 18天后用流式细胞仪和聚合酶链式反应进行检测 移植此外，幼稚(CD45RA+和CD62L+)CD4+和 CD3+8+细胞是T细胞的主要表型 这一次嫁接随后停滞不前，但坚持 10%至15%的PBL持续133天的无细胞毒性T细胞反应 在这种程度的动物中只检测到抗小鼠CD24 持久的、长寿的、高水平的多基因标记 包括原始T细胞在内的造血系具有重要的 干细胞基因治疗领域的启示