CAJ | 학술논문

β地中海贫血是一种因β珠蛋白基因缺陷导致的遗传性贫血性疾病,基因治疗是唯一有望治愈该病的方法.2型腺相关病毒(adeno-associated virus type 2,AAV2)是一种非致病性病毒,作为一种基因治疗载体,其应用潜力日益受到关注.目前还未见AAV2转导人早期胎肝造血细胞及其介导β珠蛋白基因在动物体内表达的实验报道.有研究表明,AAV2转导人造血干细胞的效率,因各实验室包装和纯化rAAV2的方法不同而存在差异,其中辅助病毒的污染被认为是一重要原因.制备了无辅助病毒污染的rAAV2,经体外检测其滴度,纯度及功能后,再转导人早期胎肝造血细胞,将被转导的胎肝造血细胞移植入受亚致死量剂量照射的8只BALB/C裸鼠体内,检测rAAV2介导的β珠蛋白基因在裸鼠体内的表达.结果显示:制备的无辅助病毒污染的rAAV2具有较高的滴度、纯度,并能够在体外介导β基因的表达;在8只受试BALB/C裸鼠中,RT-PCR在2只BALB/C裸鼠骨髓中检测到β珠蛋白基因的表达.提示,rAAV2能够转导人早期胎肝细胞并介导β珠蛋白基因的表达,但同时也存在表达量较低的缺点,应用于β地中海贫血的基因治疗还需要对AAV2生物学特性做深入的研究.
β지중해빈혈시일충인β주단백기인결함도치적유전성빈혈성질병,기인치료시유일유망치유해병적방법.2형선상관병독(adeno-associated virus type 2,AAV2)시일충비치병성병독,작위일충기인치료재체,기응용잠력일익수도관주.목전환미견AAV2전도인조기태간조혈세포급기개도β주단백기인재동물체내표체적실험보도.유연구표명,AAV2전도인조혈간세포적효솔,인각실험실포장화순화rAAV2적방법불동이존재차이,기중보조병독적오염피인위시일중요원인.제비료무보조병독오염적rAAV2,경체외검측기적도,순도급공능후,재전도인조기태간조혈세포,장피전도적태간조혈세포이식입수아치사량제량조사적8지BALB/C라서체내,검측rAAV2개도적β주단백기인재라서체내적표체.결과현시:제비적무보조병독오염적rAAV2구유교고적적도、순도,병능구재체외개도β기인적표체;재8지수시BALB/C라서중,RT-PCR재2지BALB/C라서골수중검측도β주단백기인적표체.제시,rAAV2능구전도인조기태간세포병개도β주단백기인적표체,단동시야존재표체량교저적결점,응용우β지중해빈혈적기인치료환수요대AAV2생물학특성주심입적연구.
β-Thalassemia is an inheritance anaemia disease due to the defect in β- globin gene. Gene therapy is considered to be the only method which could cure this disease. Adeno-associated virus type 2 (AAV2) has benn gaining more attention as a vector in human gene therapy for its non pathogenic character and broad host range. Although, the efficacy of recombinant AAV2 (rAAV2) in transducing human hematopoietic stem cells has been investigated by researchers, the results were varied from different laboratory. The view was proposed recently that it may be resulted from helper virus in their packaging system. Respecting this, the packaging system without helper virus was used to produce rAAV2. Human early fetal liver hematopoietic cells not only possess many superior peculiarity compared to hematopoietic cells of bone marrow or cord blood, but also the inherent β-globin gene in the cells is not expressed. Studies on the AAV2 transduction of human fetal liver hematopoietic cells and mediated expression of β- globin gene in vivo were performed and the potential role of AAV2 in β-thalassemia gene therapy was analyzed. The rAAV2 containing a normal human β-globin gene (rAAV2-β-globin) without helper virus contamination were produced. The viral titer, purity and the ability of mediating expression of β-globin gene were detected in vitro. Then, human early fetal liver hematopoietic cells were isolated and were further transducted with the rAAV2-β-globin, followed by transplantation into sublethally irradiated BALB/C nude mice to analyze the β-globin gene expression. The results showed that the high titer and purity of rAAV2-β-globin had the ability of mediating β-globin gene expression in vitro. In 8 recipient BALB/C nude mice, the β-globin gene expression were detected in the 2 mice marrow by RT-PCR. The results suggested that rAAV2 could transduce human fetal liver hematopoietic cells and mediate the β-globin gene expression in BALB/C nude mice, meanwhile the expression level of the gene was still rather low. It is necessary to perform further research on AAV2 biology before applying in β-thalassemia gene therapy.