One-step genetic correction of hemoglobin E/beta-thalassemia patient-derived iPSCs by the CRISPR/Cas9 system

Stem Cell Research & Therapy
Methichit WattanapanitchSurapol Issaragrisil

Abstract

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. 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. The hemoglobin E mutation of HbE/β-thalassemia iPSCs was seamlessly corrected by the CR...Continue Reading

References

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Citations

Sep 9, 2020·BioDrugs : Clinical Immunotherapeutics, Biopharmaceuticals and Gene Therapy·Karine Sii-FeliceEmmanuel Payen
Mar 25, 2020·Bioscience Reports·Matthew P HirakawaKimberly S Butler
Jun 7, 2019·Disease Models & Mechanisms·Maria Georgomanoli, Eirini P Papapetrou
Nov 8, 2019·Blood Advances·Jean-Yves MétaisMitchell J Weiss
Sep 29, 2020·Biologics : Targets & Therapy·Annette d'Arqom
Jun 14, 2019·Stem Cells International·Methichit Wattanapanitch
Nov 27, 2019·World Journal of Stem Cells·Maria Teresa ValentiDonato Zipeto
Feb 3, 2021·Journal of Clinical Medicine·Giacomo Frati, Annarita Miccio
Nov 18, 2020·Stem Cell Research & Therapy·Mohsen EbrahimiMaryam Farzaneh
Nov 13, 2020·Stem Cell Research & Therapy·Ratchapong NetsrithongMethichit Wattanapanitch
Aug 11, 2020·Molecular Therapy. Methods & Clinical Development·Martijn P T ErnstW W M Pim Pijnappel

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Methods Mentioned

BETA
biopsies
Assay
PCR
flow cytometry
Protein Assay
transfection
genetic modifications

Software Mentioned

ssODN
FACSCalibur
CELLQuest
BLAST
ImageQuant
Crispr Design Tool

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