Urbinati Fabrizia, Lotti Francesco, Facchini Giulia, Montanari Monica, Ferrari Giuliana, Mavilio Fulvio, Grande Alexis
Department of Biomedical Sciences, University of Modena and Reggio Emilia, 41100 Modena, Italy.
Hum Gene Ther. 2005 May;16(5):594-608. doi: 10.1089/hum.2005.16.594.
Transplantation of genetically modified hematopoietic stem cells (HSCs) has therapeutic potential for a variety of blood genetic disorders. Engraftment of HSCs, however, requires toxic myeloablative treatments, which render this approach questionable for non-life-threatening disorders. A potential alternative is the use of transgenes, which allows positive selection of HSCs in vivo. We used retroviral vectors to express a truncated derivative of the erythropoietin receptor (tEpoR) in murine and human hematopoietic cells. Murine HSCs expressing tEpoR at different levels (1500 to 13,000 receptors/cell) acquire a competitive repopulation capacity in vivo upon transplantation into fully or partially myeloablated co-isogenic mouse recipients. Long-term analysis of transplanted mice showed that expression of tEpoR at paraphysiological levels (approximately 1500 receptors/cell) has no effect on steady-state hematopoiesis and induces no further expansion of transduced cells after the engraftment period. Human cord blood-derived CD34+ stem/progenitor cells transduced with a lentiviral vector expressing tEpoR expand their clonogenic capacity in vitro, and significantly increase their marrow repopulation capacity upon xenotransplantation into sublethally irradiated NOD-SCID mice, with no alteration in their phenotype, survival, and differentiation properties. These data indicate that expression of tEpoR is an effective strategy to promote selective engraftment of genetically modified HSCs upon transplantation in both myeloablative and nonmyeloablative conditions, without the use of toxic drugs for selection.
基因改造造血干细胞（HSCs）移植对多种血液遗传疾病具有治疗潜力。然而，HSCs的植入需要进行毒性清髓治疗，这使得这种方法对于非危及生命的疾病存在疑问。一种潜在的替代方法是使用转基因，它能在体内对HSCs进行阳性选择。我们使用逆转录病毒载体在小鼠和人类造血细胞中表达促红细胞生成素受体的截短衍生物（tEpoR）。将表达不同水平tEpoR（1500至13000个受体/细胞）的小鼠HSCs移植到完全或部分清髓的同基因小鼠受体中后，它们在体内获得了竞争性的再增殖能力。对移植小鼠的长期分析表明，在生理水平（约1500个受体/细胞）表达tEpoR对稳态造血没有影响，并且在植入期后不会诱导转导细胞的进一步扩增。用表达tEpoR的慢病毒载体转导的人脐带血来源的CD34+干/祖细胞在体外扩展了其克隆形成能力，并且在亚致死照射的NOD-SCID小鼠异种移植后显著提高了其骨髓再增殖能力，而其表型、存活和分化特性没有改变。这些数据表明，tEpoR的表达是一种有效的策略，可促进在清髓和非清髓条件下移植时基因改造的HSCs的选择性植入，而无需使用毒性药物进行选择。
