地中海贫血是一种以血红蛋白产生减少为特征的遗传性血液疾病。严重贫血会损害器官,严重威胁生命安全。目前,骨髓来源的造血干细胞(HSC)的同种异体移植是一种有希望的治疗地中海贫血的方法。然而,免疫排斥和缺乏HLA匹配的供体限制了其临床应用。近年来,人诱导多能干细胞(hiPSCs)技术为基于自体细胞的治疗提供了前景,因为它可以避免上述免疫学问题。
在本研究中,我们建立了一个来自胎儿羊膜细胞的新hiPSCs系,HBB基因具有纯合β41-42(TCTT)缺失突变,HBA2基因具有杂合Westmead突变(C>G).我们设计了一个CRISPR-Cas9来靶向这些偶然的突变并纠正它们。通过全外显子组捕获测序进行基因校正的脱靶分析。通过畸胎瘤形成和成红细胞分化测定分析校正的hiPSC。
通过CRISPR/Cas9介导的同源定向修复用线性化供体DNA校正这些突变。通过序列验证hiPSC的校正。校正的hiPSC保持正常的多能性。此外,它们可以分化为造血祖细胞,这证明它们保持着多谱系分化的潜力。
我们设计了sgRNA,并证明这些促进CRISPR-Cas9基因组编辑系统的sgRNA可用于纠正患者来源的hiPSC中并发的α-和β-地中海贫血。在未来,这些校正的hiPSCs可用于并发α-和β-地中海贫血患者的自体移植。
Thalassemia is a genetic blood disorder characterized by decreased hemoglobin production. Severe anemia can damage organs and severe threat to life safety. Allogeneic transplantation of bone marrow-derived hematopoietic stem cell (HSCs) at present represents a promising therapeutic approach for thalassemia. However, immune rejection and lack of HLA-matched donors limited its clinical application. In recent years, human-induced pluripotent stem cells (hiPSCs) technology offers prospects for autologous cell-based therapy since it could avoid the immunological problems mentioned above.
In the present study, we established a new hiPSCs line derived from amniotic cells of a fetus with a homozygous β41-42 (TCTT) deletion mutation in the HBB gene and a heterozygous Westmead mutation (C > G) in the HBA2 gene. We designed a CRISPR-Cas9 to target these casual mutations and corrected them. Gene-corrected off-target analysis was performed by whole-exome capture sequencing. The corrected hiPSCs were analyzed by teratoma formation and erythroblasts differentiation assays.
These mutations were corrected with linearized donor DNA through CRISPR/Cas9-mediated homology-directed repair. Corrections of hiPSCs were validated by sequences. The corrected hiPSCs retain normal pluripotency. Moreover, they could be differentiated into hematopoietic progenitors, which proves that they maintain the multilineage differentiation potential.
We designed sgRNAs and demonstrated that these sgRNAs facilitating the CRISPR-Cas9 genomic editing system could be applied to correct concurrent α- and β-thalassemia in patient-derived hiPSCs. In the future, these corrected hiPSCs can be applied for autologous transplantation in patients with concurrent α- and β-thalassemia.