上海交通大学学报（医学版）

›› 2011, Vol. 31 ›› Issue (1): 9-.doi: 10.3969/j.issn.1674-8115.2011.01.003

• 论著（基础研究） • 上一篇    下一篇

2型重组腺相关病毒介导的β-地中海贫血基因治疗实验

田 晶1,2, 王 峰1,2, 薛金凤1,2, 赵 霏1,2, 钟 梅3, 宋柳江1,2, 谭孟群1,2   

  1. 1.中南大学 湘雅医学院生理学系血液生理研究室, 长沙 410078；2.深圳湘雅生物医药研究院, 深圳 518057；3.广州南方医科大学南方医院妇产科, 广州 510515
  • 出版日期:2011-01-28 发布日期:2011-02-01
  • 通讯作者: 谭孟群, 电子信箱: mqtan26@hotmail.com。
  • 作者简介:田 晶（1980—）, 女, 博士生；电子信箱: jingtian117@yahoo.com.cn。
  • 基金资助:

    国家科学自然基金（30470743, 30971299）；国家“重大新药创制”科技重大专项(2009ZX09503-019)；教育部高等学校博士点基金（20040533031）

Recombinant adeno-associated virus 2-mediated gene therapy for β-thalassemia

TIAN Jing1,2, WANG Feng1,2, XUE Jin-feng1,2, ZHAO Fei1,2, ZHONG Mei3, SONG Liu-jiang1,2, TAN Meng-qun1,2   

  1. 1.Laboratory of Blood Physiology, Department of Physiology, Central South University Xiangya School of Medicine, Changsha 410078, China；2.Shenzhen Xiangya Institute of Biomedicine, Shenzhen 518057, China；3.Department of Obstetrics and Gynecology, Nanfang Hospital, South Medical University, Guangzhou 510515, China
  • Online:2011-01-28 Published:2011-02-01
  • Supported by:

    National Natural Science Foundation of China, 30470743, 30971299；Special Foundation for State Major New Drug Research Program of China, 2009ZX09503-019；Foundation for University Doctorate from The Ministry of Education of China, 20040533031

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摘要：

目的 探讨2型重组腺相关病毒（rAAV2）能否有效转染重型β-地中海贫血患者造血细胞，通过体外途径基因治疗地中海贫血。方法 6只BALB/c裸小鼠分为转染组（n=4）和未转染组（n=2），经X射线照射后，分别移植入经rAAV2 β-珠蛋白病毒转染（MOI=50）或未转染的β41-42/β654杂合子型重型β-地中海贫血流产胎儿造血细胞，转染后第28天和第70天分别处死rAAV2转染小鼠（n=2）和未转染受体小鼠（n=1）。采用 RT-PCR、等位基因特异性PCR（ASPCR）法检测rAAV2介导的人β-珠蛋白基因在小鼠骨髓中的表达，并以高压液相色谱法量化分析受体小鼠外周血中人β-珠蛋白肽链的水平。结果 ①RT-PCR于所有受体小鼠样本中均可检测到人β-actin和人β-珠蛋白基因的表达。②ASPCR检测中，β41-42突变基因稳定表达于所有受体小鼠，β654突变基因仅在移植后第70天受体小鼠样本中被检测到；移植后第28天，rAAV2转染及未转染小鼠体内均可检测到主要来自于β654突变基因的正常β-珠蛋白基因表达；但至移植后第70天，仅rAAV2转染小鼠骨髓样本中仍可检测到rAAV2-β-globin载体介导的正常β-珠蛋白基因表达。③移植后第70天处死的转染小鼠外周血中人β链/α链分别为0.328和0.325，相对未转染组0.135的比值，两者的增加百分比高达144.78%和142.54%。结论 经rAAV2介导基因修饰后的重型β-地中海贫血患者造血细胞在体内可长期稳定表达正常人β-珠蛋白基因，而其分化产生的外周血人红系细胞内β-珠蛋白肽链的合成增加。

关键词: 重组腺相关病毒, β-地中海贫血, 人造血细胞, 基因治疗

Abstract:

Objective To investigate the transfection of recombinant adeno-associated virus 2 (rAAV2) in human hematopoietic stem cells of patients with βthalassemia, and explore the feasibility of ex vivo gene therapy for β-thalassemia. Methods Six BALB/c nude mice pretreated with X-ray were divided into rAAV2-transfected group (n=4) and mock-transfected group (n=2). Isolated human hematopoietic cells from fetus with β-thalassemia major of β41-42/β654 heterozygote were infected or mock infected with rAAV2-β-globin (MOI＝50), and were transplanted into BALB/c nude mice. After transfection for 28 d and 70 d, mice were sacrificed, RT-PCR and allele-specific PCR (ASPCR) were applied to detect the expression of rAAV2-mediated β-globin gene in mouse bone marrow cells, and the levels of β-globin chains in peripheral blood of recipient mice were quantified by HPLC. Results ①The expression of human β-actin and β-globin gene was detected in all recipient mice by RT-PCR. ②By ASPCR, the expression of β41-42 was found in all recipient mice, while β654 in recipient mice on day 70. There was expression of normal β-globin gene mainly from β654 gene in both groups of mice on day 28. However, on day 70,  rAAV-2 mediated normal β-globin gene expression was found only in transfected mice.  ③The human β/α in peripheral blood of transfected mice were 0.328 and 0.325 on 70 d, 144.78% and 142.54% higher than that of mock-transfected mice (0.135). Conclusion rAAV2 vectors genetically modified hematopoietic cells of patients with β-thalassemia major could mediate long-term stable expression of normal β-globin gene in vivo, with significantly elevated expression of β-chains in human erythroid cells in peripheral blood.

Key words: recombinant adeno-associated virus, β-thalassemia, human hematopoietic cells, gene therapy