TRANSFER AND EXPRESSION OF HUMAN BETA GLOBIN GENES

该项目的长期目标是改善或治愈镰状细胞 疾病的逆转录病毒基因转移的正常功能的人β或 γ球蛋白基因进入造血祖细胞（HPC），包括 这些疾病患者的干细胞。逆转录病毒载体， 这些珠蛋白基因和它们的控制元件，如基因座控制， 区域（LCR）将用于从骨髓或骨髓中扩增人HPC。 收集外周血祖细胞（PBPC）。最终目标是 通过自体移植治疗患者， 镰状细胞病患者，转导这些细胞， 高水平的γ或β珠蛋白表达，然后重新注入基因- 将纠正后的细胞输回患者体内。在以下方面取得了进展： 过去五年的研究工作包括：（1）β和γ珠蛋白基因的构建 含有稳定地传递到靶细胞中的逆转录病毒载体 小鼠HPC;和（2）用于转移和表达人HPC的条件 基因，如人鼠HPC;和（2）条件， 转移和表达人基因如人鼠HPC;和 (2)转移和表达人类基因的条件，如 在小鼠和人HPC中人多药耐药（MDR）基因。在 在这项资助的研究中，改进的含人类珠蛋白基因的载体将 在小鼠和人HPC中构建和测试，以及人珠蛋白基因- 将构建含有载体的载体并在鼠和人HPC中进行测试， 以及更有效的转移和表达这些基因的方法 同时保持它们的长期繁殖能力; 这些方法将包括使用隔离的HPC亚群， 长期骨髓培养和HPC的反复转导。此外，本发明还提供了一种方法， 将人MDR cDNA加入到含有珠蛋白基因的载体中， 提供了一种可用于富集珠蛋白基因的选择标记， 转导的HPC在体外和体内。条件有利于 珠蛋白基因转导的HPC的植入和表达 没有骨髓消融将寻求以及最后，当我们有 合适的含人珠蛋白基因的载体和培养条件 对于它们在小鼠和人HPC中的转导和表达，我们计划 设计并启动镰状细胞患者的I期临床试验， 测试逆转录病毒珠蛋白基因转移作为 治疗这些疾病的方法。

The long-term goal of this project is to ameliorate or cure sickle cell disease by retroviral gene transfer of normal functioning human beta or gamma globin genes into the hematopoietic progenitor cells (HPC) including stem cells of patients with these disorders. Retroviral vectors containing these globin genes and their control elements such as the locus control region (LCR) will be used to transduce human HPC from bone marrow or peripheral blood progenitor cell (PBPC) harvests. Ultimately, the goal is to cure the patients by autotransplantation by harvesting of HPC from patients with sickle cell disease, transducing these cells to restore high-level gamma or beta globin expression, and then re-infusing the gene- corrected cells back into the patients. Progress has been made over the past five years in: (1) the construction of beta and gamma globin gene containing retroviral vectors that are stably transmitted into target murine HPC; and (2) the conditions for transferring and expressing human genes such as the human murine HPC; and (2) the conditions for transferring and expressing human genes such as the human murine HPC; and (2) the conditions for transferring and expressing human genes such as the human multiple drug resistance (MDR) gene in murine and human HPC. In studies in this grant, improved human globin gene-containing vectors will be constructed and tested in murine and human HPC, and human globin gene- containing vectors will be constructed and tested in murine and human HPC, and more efficient methods of transferring and expressing these genes while maintaining their long term repopulating ability will be explored; these methods will include the use of isolated sub-populations of HPC, long-term marrow culture and repeated transduction of HPC. In addition, the human MDR cDNA will be added to globin gene-containing vectors to provide a selectable marker that can be used to enrich for globin gene- transduced HPC in vitro and in vivo. Conditions which favor the engraftment and expression and expression of globin gene-transduced HPC without marrow ablation will be sought as well Lastly, when we have appropriate human globin gene-containing vectors and culture conditions for their transduction and expression in murine and human HPC, we plan to design and initiate phase 1 clinical trials in sickle cell patients to test the safety and efficacy of retroviral globin gene transfer as an approach to the treatment of these disorders.