In Vivo Analysis of Hematopoietic Stem Cell Behavior in Cynomolgus Monkeys

造血干细胞具有自我更新和多向分化的能力，是基因治疗的理想靶点。逆转录病毒载体广泛用于HSC基因治疗。然而，HSC基因标记和治疗的最初人体试验显示，与在小鼠实验中观察到的高得多的效率相比，用逆转录病毒载体将基因转移到人HSC中的效率非常低。人HSC的更静止的性质和其上的较低密度的逆转录病毒受体阻碍了逆转录病毒载体的有效基因转移。由于非人灵长类动物在包括HSC生物学在内的各个方面与人类具有显着的相似性，因此它们的模型被认为对于评估和改善向人类HSC的基因转移非常重要。我们已经建立了用于自体HSC移植的非人灵长类食蟹猴模型。使用该模型，相对高水平（5 - 15%）的基因修饰的造血祖细胞已被证明是一种有效的方法。 ...更多信息 在将基因转移到HSC及其自体移植后进行。通过改善离体转导条件，例如在转导过程中将Flt-3配体和特异性纤连蛋白片段（CH-296）引入离体培养物中以及使用用巨猿白血病病毒包膜假型化的逆转录病毒载体，这已经成为可能。我们的新策略，使用选择性放大器基因（也指定的分子生长开关），现在正在测试，以进一步增加部分基因修饰的细胞使用非人灵长类动物model.Although，在小鼠中，整个造血可以重建由一个或几个造血干细胞，在较大的动物造血克隆性的数据很少。上述相对高水平的稳定逆转录病毒基因转移使我们能够进行造血的克隆分析。我们目前正在使用反向PCR来表征特定的逆转录病毒标记的克隆（通过独特的插入位点侧翼的前病毒），随着时间的推移，有助于造血。少

Hematopoietic stem cells (HSCs) are desirable targets for gene therapy because of their self-renewal and multilineage differentiation ability. Retroviral vectors are extensively used for HSC gene therapy. However, the initial human trials of HSC gene marking and therapy showed that the gene transfer efficiency into human HSCs with retroviral vectors was very low in contrast to the much higher efficiency observed in murine experiments. The more quiescent nature of human HSCs and the lower density of retroviral receptors on them hindered the efficient gene transfer with retroviral vectors. Since nonhuman primates have marked similarity to humans in all aspects including the HSC biology, their models are considered to be important to evaluate and improve gene transfer into human HSCs. We have established a nonhuman primate cynomolgus macaque model for autologous HSC transplantation. Using this model, relatively high levels (5-15%) of gene-modified hematopoietic progenitor cells has been a … More chieved after gene transfer into HSCs and their autologous transplantation. This has been made possible by improving ex vivo transduction conditions such as introduction of Flt-3 ligand and specific fibronectin fragment (CH-296) into ex vivo culture during transduction and the use of retroviral vectors pseudotyped with the gibbon ape leukemia virus envelope. Our novel strategy of using the selective amplifier gene (also designated the molecular growth switch) is now being tested to further increase the fraction of gene-modified cells using the nonhuman primate model.Although, in mice, the entire hematopoiesis can be reconstituted by a single or a few HSCs, little data about clonality of hematopoiesis in larger animals have been available. Relatively high levels of stable retroviral gene transfer described above have allowed us to conduct clonal analysis of hematopoiesis. We are currently using inverse PCR to characterize the specific retrovirally-marked clones (by the unique insertion site flanking the provirus) contributing to hematopoiesis over time. Less