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录因子转移到可以激活患者正常γ -珠蛋白基因的载体来尝试。我们建议1)研究能够保护转移基因免受整合到干细胞基因组位置的负面影响的因素。具体来说，测试鸡β基因座结构域的边界元素是否在哺乳动物细胞中起绝缘体的作用；寻找人类-珠蛋白位点的绝缘体元件；调查来自其他基因座的假定绝缘子；在转导的小鼠造血干细胞移植后，在体内测试这些元素。2)为病毒载体开发最佳的珠蛋白基因表达盒。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的LCR盒；在正常小鼠和地中海贫血或镰状细胞小鼠中制备携带最佳珠蛋白基因表达盒的病毒载体并测试其干细胞转导后的功能。3)利用我们克隆的两种新的转录激活因子γ和epsilon基因表达，检验反激活基因治疗β链血红蛋白病的可行性；检测其过表达是否影响正常小鼠造血功能；研究它们是否能在携带人β位点YACs或muLCR/Agamma基因重组体的转基因小鼠中反激活人珠蛋白基因；并测试携带这些转录因子基因编码的病毒载体的干细胞转移是否能治愈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.