联合应用phiC31整合酶与微环载体对β654地中海贫血小鼠进行基因治疗

β地中海贫血（β地贫）是常见和严重的遗传性血液病，迄今尚无有效的治疗措施。目前常用于基因治疗的慢病毒载体虽具有转移效率高等优势，但存在因外源基因随机插入而导致的安全隐患及表达不稳定等缺点。来源于链霉菌噬菌体的phiC31整合酶可介导外源基因位点特异性整合，而微环载体可在质粒载体上删除细菌元件和抗性基因从而提高外源基因表达。本课题利用phiC31整合酶系统位点特异性整合的优势及微环载体高效表达的特性制备β654地贫新型基因治疗载体。首先，优化phiC31整合酶系统中attB序列的位置、方向、数目及串联方式。其次，运用诱导型启动子体外生成血红蛋白微环载体，导入β654地贫小鼠造血干细胞对基因缺陷进行修饰。再次，将修饰后的干细胞输入β654地贫小鼠。综合应用分子生物学、细胞生物学及血液学等技术，分别在细胞和个体水平阐明新型基因治疗载体的安全性、稳定性与有效性，为β地贫的治疗提供理论和技术基础。

Beta thalassemia is a common and severe inherited hemotalogic disease. Currently, there is no effective treatment in this hematonosis. The commonly used lentiviral vectors in gene therapy has been proven to be effecient in tranfer infection. However, it has obvious disadvantages in the safety and unstability due to the random insertion in the host genome. The phiC31 integrase system is a phage-derived system that can mediate the recombination between the attB site in streptomycete genome and the attP site in phage genome. This integrase had been reported to apply in the gene therapy of hemophilia and vascellum disease for its site-specific characteristics in exogenous gene expression. While, the expression level of exogenous gene mediated by phiC31 integrase was unstable due to the positional effect of site specific insertion in host genome. Minicircle vector could enhance the expression level of exogenous gene by deleting elements of plasmid and antibiotic gene. Our study will use the combined techniques of phiC31 integrase system and minicircle vector to both improve the expression efficiency in constructing vectors and the safety of gene therapy of thalassemic beta 654 mice. First of all, we will optimize the phiC31 by adjusting the position,direction,number and linkage of attB sequence. We then compare the exogenous gene expression efficiency between the optimized and unoptimized vector. Second, we will construct the human beta-hemoglobin minicirle vector by inducing the promoter of Cre recombinase. Third, we will use the combined techniques of phiC31 and minicircle to construct the new vector and transfected into the hemopoietic stem cells(HSCs). Fourth, modified HSCs will be purified by flow cytometry and then injected into the tail vein of mice with beta 654 thalassemia. Finally, the security, stability and efficiency of constructed vectors with phiC31 integrase and minicircle in gene therapy will be examined by molecular biologic,cell biologic and hematologic approaches. Our study would provide a new tool in the gene therapy of beta thalassemia.