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: 6453770
• 负责人: M ROSENZWEIG
• 金额: $ 11.11万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6453770
• 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

优化造血的收获和转导 干细胞对于成功的干细胞基因疗法至关重要 评估了涉及FLT-3和G-CSF的新方案的实用性 动员外周血干细胞和逆转录病毒 使用造血生长因子转导的转导 记者基因鼠CD24进入非人类的造血干细胞 灵长类猕猴用FLT-3配体（200 mg/kg）处理 和G-CSF（20 mg/kg）持续7天，自体CD34+外周血 用白细胞术CD34+细胞收获的干细胞与 基于MFG的逆转录病毒载体，使用4每天编码鼠CD24 在FLT-3配体存在下离心的转导 （100 ng/ml），SCF（100 ng/ml）和Pixy（100 ng/ml）在无血清培养基中 当天，动物在共辐照（400 cgy） 在骨骼中依次监测移植植入 骨髓和血液都使用多参数浮动W细胞仪和 半定量DNA PCR我们的数据表明成功，并且 多种造血谱系中的持续植入，包括 46和117天的粒细胞，单核细胞和B和T淋巴细胞 移植后分别为75％和6％的粒细胞 在细胞表面峰值的体内水平上表达MCD24抗原 遗传改性的外周血淋巴细胞（PBL）接近60％ 如流式细胞术和PCR 18天评估 此外，移植还原（CD45RA+和CD62L+）CD4+和 CD3+ 8+细胞是检测到的T细胞的主要表型 这次植入随后静止了，但一直持续 在10％至15％的PBL中，持续133天毒性T细胞反应 仅在一只动物中检测到针对鼠CD24 持续的长寿命长，高水平的基因标记 造血谱系，包括幼稚的T细胞，具有重要的 对干细胞基因治疗领域的影响