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地中海贫血的基因治疗方法。这将通过开发将伽马珠蛋白基因转移到患者干细胞中的载体或将转移转录因子的载体来尝试，这些转录因子可以激活患者的正常伽马珠蛋白基因。我们建议1)研究哪些因素将有利于保护被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。具体地说，测试鸡β基因座区域的边界元件是否在哺乳动物细胞中起绝缘体的作用；寻找人β珠蛋白基因座的绝缘体元件；研究其他基因座的假定绝缘体；在转导的小鼠造血干细胞移植后在体内测试这些元件。2)研制适合病毒载体的珠蛋白基因表达盒。具体地说，改进我们已经开发的表达盒，研究β基因座控制区(LCR)的结构与功能的关系，并利用这一知识生产最佳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、地中海贫血或镰状细胞小鼠进行干细胞转导后测试它们的功能。3)用我们克隆的两个新的转录激活子

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.