Hematti Peiman, Sellers Stephanie E, Agricola Brian A, Metzger Mark E, Donahue Robert E, Dunbar Cynthia E
Hematology Branch, NHLBI, National Institutes of Health, Bethesda, MD 20892, USA.
Blood. 2003 Mar 15;101(6):2199-205. doi: 10.1182/blood-2002-08-2663. Epub 2002 Nov 7.
Gene transfer experiments in nonhuman primates have been shown to be predictive of success in human clinical gene therapy trials. In most nonhuman primate studies, hematopoietic stem cells (HSCs) collected from the peripheral blood or bone marrow after administration of granulocyte colony-stimulating factor (G-CSF) + stem cell factor (SCF) have been used as targets, but this cytokine combination is not generally available for clinical use, and the optimum target cell population has not been systematically studied. In our current study we tested the retroviral transduction efficiency of rhesus macaque peripheral blood CD34(+) cells collected after administration of different cytokine mobilization regimens, directly comparing G-CSF+SCF versus G-CSF alone or G-CSF+Flt3-L in competitive repopulation assays. Vector supernatant was added daily for 96 hours in the presence of stimulatory cytokines. The transduction efficiency of HSCs as assessed by in vitro colony-forming assays was equivalent in all 5 animals tested, but the in vivo levels of mononuclear cell and granulocyte marking was higher at all time points derived from target CD34(+) cells collected after G-CSF+SCF mobilization compared with target cells collected after G-CSF (n = 3) or G-CSF+Flt3-L (n = 2) mobilization. In 3 of the animals long-term marking levels of 5% to 25% were achieved, but originating only from the G-CSF+SCF-mobilized target cells. Transduction efficiency of HSCs collected by different mobilization regimens can vary significantly and is superior with G-CSF+SCF administration. The difference in transduction efficiency of HSCs collected from different sources should be considered whenever planning clinical gene therapy trials and should preferably be tested directly in comparative studies.
在非人类灵长类动物中进行的基因转移实验已被证明可预测人类临床基因治疗试验的成功。在大多数非人类灵长类动物研究中，在给予粒细胞集落刺激因子（G-CSF）+干细胞因子（SCF）后从外周血或骨髓中收集的造血干细胞（HSCs）已被用作靶细胞，但这种细胞因子组合一般不可用于临床，并且尚未对最佳靶细胞群体进行系统研究。在我们目前的研究中，我们测试了在给予不同细胞因子动员方案后收集的恒河猴外周血CD34(+)细胞的逆转录病毒转导效率，在竞争性再增殖试验中直接比较了G-CSF+SCF与单独使用G-CSF或G-CSF+Flt3-L的情况。在刺激细胞因子存在的情况下，每天添加载体上清液，持续96小时。通过体外集落形成试验评估的HSCs转导效率在所有5只受试动物中相当，但与G-CSF（n = 3）或G-CSF+Flt3-L（n = 2）动员后收集的靶细胞相比，在G-CSF+SCF动员后收集的靶CD34(+)细胞来源的所有时间点，体内单核细胞和粒细胞标记水平更高。在3只动物中，实现了5%至25%的长期标记水平，但仅源于G-CSF+SCF动员的靶细胞。不同动员方案收集的HSCs的转导效率可能有显著差异，G-CSF+SCF给药时转导效率更高。在规划临床基因治疗试验时，应考虑从不同来源收集的HSCs转导效率的差异，并且最好在比较研究中直接进行测试。
