ENGINEERING STEM CELLS TO CONFER PROLIFERATIVE ADVANTAGE

子宫内造血干细胞移植(IUT)通过产生造血干细胞(HSCs)有望治愈多种血液病，但IUT所能实现的供体细胞植入水平较低，限制了该疗法的应用。我们的目标是将宫内节育器的使用扩展到治疗镰状细胞性贫血等疾病。为了达到治疗这种疾病所需的高水平的供体细胞植入，这项建议的目标是使HSCs具有比正常HSCs更多的增殖优势。这项提议将检验这样一种假设，即将促红细胞生成素(EPOR)引入HSC将使这些改变的细胞对促红细胞生成素(EPO)产生反应。这将导致改变的HSCs及其后代比正常的祖细胞具有增殖优势。截断形式的EPOR(TEpoR)也将进行测试。这些tEpoR在其细胞质结构域的负调节区有缺失，比EPOR提供更强的增殖信号。胎儿HSCs将被用作靶点，因为它们比成人细胞具有增殖优势，因此容易被逆转录病毒载体转导。慢病毒载体还将测试它们修改胎儿和出生后HSCs来源的能力。EPOR基因的导入对HSCs增殖和分化的影响将通过不同的体外培养系统来确定。假设EPOR或tEpoR在HSCs上的异位表达可以使这些细胞比正常的HSCs更具竞争力，将在人胎儿造血的小鼠模型中测试经修饰的HSCs与对照HSCs。体内模型也将被用来测试EPO对修饰的HSCs扩增的影响。实验的能力将进一步确定是否使HSCs对HPO作出反应，是否会对HSCs的长期重建和多系潜能产生不利影响。

In utero hematopoietic stem cell transplantation (IUT) offers the hope of curing a number of hematological diseases by generating a state of hematopoietic stem cells (HSCs), the levels of donor cell engraftment that can be achieved by IUT are low, limiting the use of this therapy. Our aim is to extend the use of IUT to the treatment of diseases such as sickle cell anemia.. To achieve the high levels of donor cell engraftment needed to treat this disease it is the goal of this proposal to engineer HSCs to have a proliferative advantage over normal HSCs. This proposal will test the hypothesis that introduction of the erythropoietin (EpoR) into HSCs will render these altered cells responsive to erythropoietin (EPO). This will result in the altered HSCs and their progeny having a proliferative advantage over normal progenitors. Truncated forms of EpoR (tEpoR) will also be tested. These tEpoR, having deletions in the negative regulatory region of their cytoplasmic domains, deliver stronger proliferative signals, than EpoR. Fetal HSCs will be used as targets since they offer proliferative advantages over adult cells and are, therefore, susceptible to transduction by retroviral vectors. Lentiviral vectors will also be tested for their capacity to modify fetal as well as postnatal sources of HSCs. The effects of introducing the EpoR genes on the proliferation and differentiation of HSCs will be determined using various in vitro culture systems. It is hypothesized that ectopic expression of either EpoR or tEpoR expression on HSCs can make these cells more competitive than their normal counterparts, modified HSCs will be tested against control HSCs in a mouse model of human fetal hematopoiesis. The in vivo model will also be used to test the effects of EPO administration on the expansion of the modified HSCs. The ability of experiments will further determine if making HSCs responsive to HPO will have detrimental effect on the long-term reconstituting- and multi- lineage potential of HSCs.