衣壳蛋白突变的新一代腺相关病毒载体介导β-珠蛋白基因治疗地中海贫血的研究

The β-thalassemias are genetic disorders of hemoglobin synthesis characterized by deficient (β+) or absent (β0) synthesis of the β-globin subunit of hemoglobin molecule. Adeno-associated viruses (AAV) are widely spread throughout the human population, yet no disease has been associated with infection. This fact, together with the ability to mediate long term exogenous gene expression, has made AAV a serious contender in gene therapy. However, their transduction efficiency in hematopoietic stem cells (HSCs) has been reported to be low, which is the key problem to utilize it in gene therapy of β-Thalassemia. In this subject, firstly, we will introduce different point mutations into AAV capsid protein by directed gene mutagenesis followed by evaluating the transduction efficiency of the vector containing different point mutations and mutation combinations in HSCs. Secondly, we will infect the HSCs from the bone marrow of β-Thalassemia children using the best mutant carrying the β-globin gene, and culture a few of those infected cells in vitro to introduce erythroid differentiation, as well as evaluate the β-globin expression level in a NOD /SCID xenograft model in vivo. β-globin protein expression levels in peripheral blood of recipients will be examined by HPLC. The gene expression levels and integration analysis will be carried in the cells from the bone marrow of recipients. This research might identify the AAV mutants to be useful for the potential gene therapy of human hemoglobinopathies in general, and β - thalassemia in particular, and provide evidence for the possibility of gene therapy of β - thalassemia.

β-地中海贫血是一种以β-珠蛋白合成减少或缺失为特征的严重致死、致残性单基因遗传病，基因治疗被认为是根治该病的唯一手段，而腺相关病毒（AAV）以其无致病性、宿主范围广、介导外源基长期表达等优点被认为是基因治疗中的最佳载体。本项目拟在前期工作基础上引进点突变技术改造衣壳蛋白，用不同单突变及不同组合突变的衣壳蛋白包装纯化获得rAAV/GFP病毒，比较各突变型病毒对造血干细胞的感染效率，筛选高效感染造血干细胞的突变型AAV，以此包装纯化获得rAAV/β-globin病毒，用该病毒感染重型β-地贫标本的造血干细胞，进行 NOD/SCID 鼠移植实验，以及诱导培养感染后的造血干细胞向红系分化， 1个月后，观察其细胞形态学变化，移植后3个月，6个月采集移植鼠标本检测β-珠蛋白合成、β-珠蛋白基因表达等多项指标，综合分析、评估该突变型AAV载体对β-地中海贫血治疗的有效性，有望该研究项目获得突破性进展。

本项目在前期工作基础上，采用分子生物学点突变技术改造腺相关病毒（AAV）衣壳蛋白，分别突变了AAV2与AAV6衣壳蛋白表面暴露的酪氨酸为苯丙氨酸包括单位点突变与双位点突变，包装纯化携带标记基因GFP的突变型重组腺相关病毒，用荧光定量PCR检测各病毒滴度，将其感染人造血干/祖细胞或K562细胞株，经流式检测比较不同突变型重组腺相关病毒的感染效率，筛选获取AAV6单突变对造血干细胞感染效率高的突变型AAV，为β-地中海贫血病的基因治疗了提供切实有效的新型AAV载体.