【作者】 赵娜；

【导师】 梁植权； 刘德培； 董文吉；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2004， 博士

【摘要】 基因治疗是指将遗传物质导入患者体内，使患者获得有益治疗的一种新型治疗方式，它是根治遗传性疾病的最直接和有效的方法。造血干细胞(hematopoietic stem cell, HSC)因具有自我更新和多谱系分化的能力成为基因治疗的最理想的靶细胞。基因治疗的基本目标是外源基因整合于HSC的染色体。 人类珠蛋白基因家族包括α-和β-两个基因簇，两簇内各功能基因呈红系组织特异性、发育阶段特异性、终产物始终维持平衡的表达。一旦平衡失调将导致地中海贫血。β-地贫的发生是由于人类β-珠蛋白基因簇中的DNA序列缺失或突变导致β-珠蛋白肽链合成不足，相对过剩的α-珠蛋白肽链自身形成四聚体，从而导致红细胞形态及功能异常。 β-地贫是人类较为常见的造血系统遗传性疾病，目前尚无疗效满意的治疗方法。β-地贫的基因治疗大多采用病毒载体介导的造血干细胞的基因转移策略。尽管病毒载体对遗传信息的传递非常有效，但是由于载体的不稳定性可导致转移基因不稳定整合和表达，而且病毒载体在转移基因的过程中可能发生的随机插入突变及可能产生的野生型重组病毒对机体危害甚大，因此寻找安全有效的载体系统是目前基因治疗领域的重要课题。为避免病毒载体的上述负面效应，在本研究中，我们引入一种新型打靶载体HCD-TV(human chromosome-derived targeting vector)。HCD-TV是一种人D，G组染色体靶向的人源载体，它利用人类D，G组染色体短臂上的序列作为打靶臂，通过同源重组将外源基因定向导入D，G组染色体短臂rDNA区，rDNA序列可以为外源基因的表达提供合适的染色质环境，最终获得外源基因的高效表达。 HCD-TV的基本骨架是细菌人工染色体(bacterial artificial chromosome, BAC)载体，它的两个打靶引导序列(targeting leading sequence)TGLS1和TGLS2分别为1.5kb和1.6kb，来源于D，G组染色体短臂，与其高度同源。打靶臂之间为正筛选基因Neo基因，打靶臂以外、TGLS2与BAC载体骨架之间为负筛选基因tk基因，Neo基因上游与TGLS2之间的NheI位点可允许外源基因的插入。携带外源基因的打靶载体转染细胞后，经过G418进行正筛选，GCV进行负筛选，所获得的细胞克隆即为外源基因定向整合克隆。 许多临床病例及实验表明胎儿型血红蛋白(fetal hemoglobin, HbF)的增更多还原

【Abstract】 Gene therapy can be defined as the modification of the genome of cells to treat or prevent diseases. It is the most direct and effective method for the treatment of hereditary disorders. Hematopoietic stem cells (HSCs) are regarded as the most ideal target cells in gene therapy because of their capacity of self-renewing (being able to give rise to literally billions of progeny cells for essentially a lifetime) and pluripotent differentiation (being able to give rise to cells of all hematopoietic and lymphoid lineages). It is an attractive treatment strategy for many genetic and hematological diseases to transfer therapeutic genes into HSCs based on integrating vectors.β-thalassemia is a kind of common hereditary disease in hematopoietic system.. Till now, no satisfactory strategies can be performed effectively in the treatment of β-thalassemia. Human globin gene family resides in different loci, α-globin gene cluster and β-globin gene cluster. The function genes in both clusters can be expressed with the characteristics of erythroid-specificity, developmental stage-specificity and final product-balance. Once the balance between α -and β -globin final products was destroyed, thalassemia is coming. β-thalassemia results from deletions or mutations in regulatory regions or coding regions of β-globin gene cluster, which leads to the descendance or cessation of the synthesis of β-globin polypeptide, and thus the aggregation of redundant α -globin peptide.Viral vectors-mediated gene transfer to HSC are mostly used in the gene therapy of β-thalassemia by now. Viral vectors are efficient but currently appear to be too dangerous for routine clinical use, because their instability might cause unstable integration and expression of target gene. What’s more, viral vectors may cause integration mutation during the procedure of gene transfer, and recombinant wild-type virus may be produced. So Gene delivery vehicles ("vectors") are the main更多还原