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Title: One-step genetic correction of hemoglobin E/beta-thalassemia patient-derived iPSCs by the CRISPR/Cas9 system
Authors: Methichit Wattanapanitch
Nattaya Damkham
Ponthip Potirat
Kongtana Trakarnsanga
Montira Janan
Yaowalak U-Pratya
Pakpoom Kheolamai
Nuttha Klincumhom
Surapol Issaragrisil
Chulalongkorn University
Faculty of Medicine, Thammasat University
Faculty of Medicine, Siriraj Hospital, Mahidol University
Keywords: Biochemistry, Genetics and Molecular Biology;Medicine
Issue Date: 26-Feb-2018
Citation: Stem Cell Research and Therapy. Vol.9, No.1 (2018)
Abstract: © 2018 The Author(s). Background: Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. We now report genetic correction of the beta hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for hemoglobin E and β-thalassemia (HbE/β-thalassemia), the most common thalassemia syndrome in Thailand and Southeast Asia. Methods: We used the CRISPR/Cas9 system to target the hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a single-stranded DNA oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and hemoglobin expression. Results: The hemoglobin E mutation of HbE/β-thalassemia iPSCs was seamlessly corrected by the CRISPR/Cas9 system. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein. Conclusions: Our study provides a strategy to correct hemoglobin E mutation in one step and these corrected iPSCs can be differentiated into hematopoietic stem cells to be used for autologous transplantation in patients with HbE/β-thalassemia in the future.
ISSN: 17576512
Appears in Collections:Scopus 2018

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