Development of gamma globin based gene therapy of hemoglobinopathies

该项目的目标是开发可用于镰状细胞病和β地中海贫血基因治疗的方法。这将通过开发将γ珠蛋白基因转移到患者干细胞中的载体或将转移可激活患者正常γ珠蛋白基因的转录因子的载体来尝试。我们建议1）研究将保护转移的基因免受整合到干细胞基因组中的位置的负面影响的元件。具体来说，测试是否边界元件的鸡β基因座域的功能作为绝缘体在哺乳动物细胞中;寻找绝缘体元件的人β珠蛋白基因座;调查推定的绝缘体从其他基因座;测试这些元素在体内移植后的转导小鼠造血干细胞。2)开发用于病毒载体的最佳珠蛋白基因表达盒。具体而言，改进我们已经开发的表达盒，研究β基因座控制区（LCR）的结构-功能关系，并利用这些知识生产最佳LCR盒;生产携带最佳珠蛋白基因表达盒的病毒载体，并在正常小鼠和地中海贫血或镰状细胞小鼠中进行干细胞转导后测试其功能。3)用我们克隆的两个新的γ和γ-D基因表达转录激活因子检测β-链血红蛋白病的反式激活基因治疗的可行性，检测它们的过表达是否影响正常小鼠的造血功能，在携带人β位点YAC或muLCR/A γ基因重组体的转基因小鼠中检测它们是否能反式激活人珠蛋白基因;并测试携带这些转录因子的基因编码的病毒载体的干细胞转移是否治愈了β地中海贫血或镰状细胞小鼠的细胞缺陷。

The objective of this project is to develop approaches that can be used for gene therapy f sickle cell disease and beta thalassemia. This will be attempted with development of vectors which will transfer the gamma globin genes into the patient' stem cells or vectors that will transfer transcriptional factors which can activate the normal gamma globin genes of the patients. We propose to 1) investigate elements that will pro protect the transferred genes from negative effects of the position of integration into the stem cell genome. Specifically, test whether the border elements of the chicken beta locus domain function as insulators in mammalian cells; search for insulator elements of the human beta globin locus; investigate putative insulators from other loci; test these elements in vivo following transplantations of transduced murine hemopoietic stem cells. 2) Develop optimal globin gene expression cassettes for viral vectors. Specifically, improve the expression cassettes we have already developed, investigate the structure-function relationships of the beta locus control region (LCR) and use this knowledge to produce optimal LCR cassettes; produce viral vectors carrying the optimal globin gene expression cassettes and test their function following stem cell transduction in normal mice and in thalassemic or sickle cell mice. 3) Test the feasibility of transactivation gene therapy of the beta chain hemoglobinopathies using the two new transcription activators of gamma and epsilon gene expression we have cloned; test whether their over- expression affects the normal murine hematopoiesis; investigate whether they can transactivate the human globin genes in transgenic mice carrying the human beta locus YACs or muLCR/Agamma gene recombinants; and test whether stem cell transfer of viral vectors carrying the genes codes for these transcriptional factors cure the cellular defects of the beta thalassemic or sickle cell mice.