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

造血干细胞的收获和转导的优化 干细胞是成功的干细胞基因治疗的关键， 评估了一种新的方案，包括flt-3和G-CSF的效用 外周血干细胞动员和逆转录病毒 使用造血生长因子进行转导， 报告基因，鼠CD 24，进入非人造血干细胞 灵长类恒河猴用flt-3配体（200 mg/kg）处理 和G-CSF（20 mg/kg）7天，以及自体CD 34+外周血 通过白细胞去除术收获的干细胞CD 34+细胞用 基于MFG的编码鼠CD 24的逆转录病毒载体，每天使用4次 在FLT-3配体存在下离心转导 (100无血清培养基中的SCF（100 ng/ml）和PIXY（100 ng/ml 在给药当天对动物进行亚致死剂量辐照（400 cGy）。 在骨中连续监测移植物植入 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的， 在多种造血谱系中持续植入，包括 46天和117天时的粒细胞、单核细胞以及B和T淋巴细胞 移植后分别有75%和6%的粒细胞， 在细胞表面表达mCD 24抗原，体内峰值水平为 转基因外周血淋巴细胞（PBL）接近60% 如在18天后通过流式细胞术和PCR两者评估的， 此外，初始（CD 45 RA+和CD 62 L+）CD 4+和 CD 3 +8+细胞是T细胞的主要表型， 这一次，植入随后达到稳定状态，但仍持续， 10 - 15%的PBL持续133天细胞毒性T细胞应答 仅在一只动物中检测到抗鼠CD 24的抗体。 持久的长寿命，高水平的基因标记的多种 造血谱系，包括幼稚T细胞，具有重要的 干细胞基因治疗领域的意义

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