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: 6591324
• 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: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6591324
• 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

造血细胞收获和转导的优化 干细胞对于成功的干细胞基因治疗至关重要 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 使用造血生长因子进行转导以引入 报告基因，鼠CD24，进入非人类的造血干细胞 用 flt-3 配体（200 毫克/千克）处理灵长类恒河猴 和 G-CSF (20 mg/kg) 7 天以及自体 CD34+ 外周血 通过白细胞去除术收获的干细胞 CD34+ 细胞被转导 基于 MFG 的逆转录病毒载体编码鼠 CD24，每天使用 4 在 flt-3 配体存在下通过离心进行转导 (100 ng/ml)、SCF (100 ng/ml) 和 PIXY (100 ng/ml)，溶于无血清培养基 在当天对动物进行亚致死照射（400 cGy） 移植 在骨中连续监测植入情况 使用多参数流式细胞术和 半定量 DNA PCR 我们的数据表明成功且 在多种造血谱系中持续植入，包括 46 和 117 天时的粒细胞、单核细胞以及 B 和 T 淋巴细胞 移植后粒细胞分别为 75% 和 6%， 在细胞表面表达 mCD24 抗原 体内峰值水平 转基因外周血淋巴细胞（PBL）接近60% 术后 18 天通过流式细胞术和 PCR 进行评估 移植 此外，初始（CD45RA+ 和 CD62L+） CD4+ 和 CD3+8+ 细胞是检测到的 T 细胞的主要表型 这次植入随后趋于稳定，但持续在 133 天无细胞毒性 T 细胞反应，PBL 介于 10% 至 15% 之间 仅在一只动物中检测到针对小鼠 CD24 的这种程度 多个持久的、长期的、高水平的基因标记 造血谱系，包括初始 T 细胞，具有重要的作用 对干细胞基因治疗领域的影响