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

Abstract

Background 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. Methods 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. Results 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. Conclusions 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.

Induced pluripotent stem cell-derived hematopoietic stem and progenitor cells: potential, challenges, and future perspectives

“…It has been mainly classified into 2 different types, α-and β-thalassemia, where α-thalassemia is usually due to a deletion within the α-globin gene, while β-thalassemia is usually caused by non-deletional mutations [78]. The genetic correction of concurrent α-and β-thalassemia in patient-derived iPSCs by the application of the CRISPR/Cas9 system was presented by Li et al in 2022 [79]. CRISPR/Cas9-mediated HDR with linearized donor DNA was conducted.…”

CRISPR/Cas9 in the treatment of -thalassemia

“…Lingli Li et al's used CRISPR-Cas9 technology to genetically correct pluripotent stem cells derived from α-and β-thalassemia patients [8]. CRISPR-Cas9 technology has successfully repaired the CD41/42 (-CTTT) mutation in the HBB gene.…”