利用CRISPR/Cas9基因编辑技术治疗血红蛋白H病的实验研究

血红蛋白H病（HbH病）是α-地中海贫血（地贫）的常见类型，其特征是α-珠蛋白链合成不足，β-珠蛋白链过剩形成HbH包涵体，导致溶血和无效造血，目前治疗手段有限。本课题组前期应用CRISPR/Cas9和小RNA干扰技术对正常人和β-地贫患者CD34+细胞进行研究，均能下调BCL11A基因表达，显著增加胎儿血红蛋白合成。本课题拟以正常人及HbH病患者为研究对象，采用CRISPR/Cas9技术编辑CD34+细胞β-珠蛋白基因的启动子区，从而靶向下调β-珠蛋白基因表达，通过qPCR法、高效液相法检测珠蛋白基因和珠蛋白链表达量，电镜观察HbH包涵体，评估α与β-珠蛋白链比例失衡及溶血的改善情况，同时评估脱靶率和对红系分化的影响；将基因编辑后的人CD34+细胞移植至NSG小鼠中，评估基因编辑的长期有效性。本课题为HbH病的基因治疗提供实验和理论基础。

Hemoglobin H disease(HbH disease) is a common type of α-thalassemia, which is characterized by deficiency of α-globin chain and excess of β-globin chain. The imbalance between α-globin and β-globin chain results in the formation of HbH inclusion body which leads to hemolysis and ineffective hematopoiesis. Current treatment is limited. We found that the expression of the BCL11A gene was down-regulated by CRISPR/Cas9 and small interfering RNA technologies to increase the expression of fetal hemoglobin in the CD34+ cells from normal donors and patients with β-thalassemia. In this study, we will edit the promoter region of β-globin gene in CD34+ cells from normal donors and patients with HbH disease by CRISPR/Cas9 for the purpose of downregulation of β-globin gene. The expression levels of globin genes and globin chains will be detected by quantitative Real-time PCR (qPCR) and High Performance Liquid Chromatography(HPLC), and HbH inclusion bodies will be observed by electron microscopy to evaluate the improvement of the imbalance with globin chains and hemolysis. Meanwhile, off-target rate and impacts on erythroid differentiation will be valuated. Subsequently, gene-edited human CD34+ cells are transplanted into mice to assess the long-term effectiveness. The study will provide a experimental and theoretical basis for gene therapy of HbH disease.