利用诱导干细胞和基因打靶技术治疗α-地中海贫血的机制研究

α-Thalassemia is a common autosomal recessive monogenetic disease in Southern China. If both parents carry the mutated α-thalassemia gene, one in four chance of fetus would be homozygous α-thalassemia. The affected fetuses present severe anemia and hydrops, and usually die in utero at third trimester of pregnancy or early after birth. The treatment of this disease is currently prenatal diagnosis and termination of pregnancy. Induced pluripotent stem cells (also known as iPSCs) are a type of pluripotent stem cell that are generated directly from adult cells. These cells hold great promise in the field of regenerative medicine because they not only bypass the need for embryos, but can be made in a patient-matched manner, which means that each individual could have their own pluripotent stem cell line without the risk of immune rejection. Based on our grasping of the technology of establishing iPSCs, we want to do researches in the following aspects: (1) reprogramming the chorionic villus cells from fetuses diagnosed in early pregnancy as homozygous α-thalassemia to iPSCs; (2) correcting the genetic mutation in α-thalassemia iPSCs by a specific gene editing technique based on the use of CRISPR/CAS9; (3) in vivo differentiation of corrected iPSCs lines towards CD34+ hematopoietic stem cells (HSCs) and detection of α-globin gene expression; (4)Transplantation of HSCs derived from α-thalassemia iPSC treats α-thalassemia mouse. The implementation of this project will provide theoretical and laboratory evidence for the treatment of homozygous α-thalassemia, and ultimately develop a therapeutic strategy other than termination of pregnancy.

α-地中海贫血（地贫）是我国南方地区常见单基因病。双方均为基因携带者的夫妇，有四分之一机会孕育重型α-地贫胎儿,表现为极度贫血、水肿，多在妊娠晚期或出生后很快死亡，目前的治疗方法是产前诊断，对患病胎儿实施流产。诱导多能干细胞(iPSCs)技术为获得患者自身遗传背景的干细胞进行临床治疗开拓了新途径。本课题组已经掌握了iPSCs建系技术，拟在前期的工作基础上开展：（1）在妊娠早期将重型α-地贫胎儿的绒毛细胞重编程iPSCs；（2）应用CRISPR/CAS9技术对iPSCs细胞内的α-珠蛋白基因突变进行修复，获得基因修正的iPSCs；（3）将修复后的iPSCs体外定向诱导分化造血干细胞及红细胞，检测其α-珠蛋白基因表达水平的恢复；（4）在α-地贫鼠模型上使用定向诱导分化的造血干细胞进行造血干移植。本项目的实施将为重型α-地贫的临床治疗提供理论及实验依据，为患病家庭提供除胎儿流产之外的治疗选择。

α-地中海贫血（α-地贫）是我国南方地区危害严重的单基因病。目前对于此病的防控策略是产前诊断，对重型α地贫胎儿实施流产。本项目旨在探讨诱导多能干细胞(iPSCs)结合基因打靶技术治疗α地贫的可能性。本项目从产前诊断出胎儿为重型α地贫（Bart’s水肿胎）的孕妇获取羊水细胞，通过体外诱导实验，建立了稳定的Bart’s-iPSCs细胞系；应用CRISPR/CAS9技术打靶Bart’s-iPSCs，成功在α地贫基因缺失处插入HBA1与HBA2基因，并保存了基因修复的Bart’s-iPSCs细胞株。同时本项目还建立了另外一种严重α地贫HbH-Constant Spring（CS）患者来源的iPSCs细胞系，用CRISPR/CAS9技术成功修复HbH-CS-iPSCs的CS突变位点（T>C），并将修复后的HbH-CS-iPSCs诱导分化为红系造血干细胞，分化效率与未修复的HbH-CS-iPSCs无显著差异；检测表明，修复后的HbH-CS-iPSCs保持了多能干性与基因组的稳定性，并无脱靶效应。因此，本项目对iPSCs技术结合CRISPR/CAS9基因编辑技术治疗α地贫做了有益的实验研究，初步显示了此治疗方法理论上的可行性，为本项目后续深入研究如动物实验、临床试验创造了条件。本项目已发表论文17篇。