Genetic correction of concurrent α- and β-thalassemia patient-derived pluripotent stem cells by the CRISPR-Cas9 technology

清脆的 诱导多能干细胞 干细胞 生物 地中海贫血 基因组编辑 遗传学 人诱导多能干细胞 胚胎干细胞 计算生物学 基因
作者
Lingli Li,Hongyan Yi,Zheng Liu,Ping Long,Tao Pan,Yuanhua Huang,Yongsheng Li,Qi Li,Yanlin Ma
出处
期刊:Stem Cell Research & Therapy [Springer Nature]
卷期号:13 (1) 被引量:8
标识
DOI:10.1186/s13287-022-02768-5
摘要

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.
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