TY - JOUR
T1 - One-Step Biallelic and Scarless Correction of a β-Thalassemia Mutation in Patient-Specific iPSCs without Drug Selection
AU - Liu, Yali
AU - Yang, Yi
AU - Kang, Xiangjin
AU - Lin, Bin
AU - Yu, Qian
AU - Song, Bing
AU - Gao, Ge
AU - Chen, Yaoyong
AU - Sun, Xiaofang
AU - Li, Xiaoping
AU - Bu, Lei
AU - Fan, Yong
N1 - Publisher Copyright:
© 2016 The Authors
PY - 2017/3/17
Y1 - 2017/3/17
N2 - Monogenic disorders (MGDs), which are caused by single gene mutations, have a serious effect on human health. Among these, β-thalassemia (β-thal) represents one of the most common hereditary hematological diseases caused by mutations in the human hemoglobin β (HBB) gene. The technologies of induced pluripotent stem cells (iPSCs) and genetic correction provide insights into the treatments for MGDs, including β-thal. However, traditional approaches for correcting mutations have a low efficiency and leave a residual footprint, which leads to some safety concerns in clinical applications. As a proof of concept, we utilized single-strand oligodeoxynucleotides (ssODNs), high-fidelity CRISPR/Cas9 nuclease, and small molecules to achieve a seamless correction of the β-41/42 (TCTT) deletion mutation in β thalassemia patient-specific iPSCs with remarkable efficiency. Additionally, off-target analysis and whole-exome sequencing results revealed that corrected cells exhibited a minimal mutational load and no off-target mutagenesis. When differentiated into hematopoietic progenitor cells (HPCs) and then further to erythroblasts, the genetically corrected cells expressed normal β-globin transcripts. Our studies provide the most efficient and safe approach for the genetic correction of the β-41/42 (TCTT) deletion in iPSCs for further potential cell therapy of β-thal, which represents a potential therapeutic avenue for the gene correction of MGD-associated mutants in patient-specific iPSCs.
AB - Monogenic disorders (MGDs), which are caused by single gene mutations, have a serious effect on human health. Among these, β-thalassemia (β-thal) represents one of the most common hereditary hematological diseases caused by mutations in the human hemoglobin β (HBB) gene. The technologies of induced pluripotent stem cells (iPSCs) and genetic correction provide insights into the treatments for MGDs, including β-thal. However, traditional approaches for correcting mutations have a low efficiency and leave a residual footprint, which leads to some safety concerns in clinical applications. As a proof of concept, we utilized single-strand oligodeoxynucleotides (ssODNs), high-fidelity CRISPR/Cas9 nuclease, and small molecules to achieve a seamless correction of the β-41/42 (TCTT) deletion mutation in β thalassemia patient-specific iPSCs with remarkable efficiency. Additionally, off-target analysis and whole-exome sequencing results revealed that corrected cells exhibited a minimal mutational load and no off-target mutagenesis. When differentiated into hematopoietic progenitor cells (HPCs) and then further to erythroblasts, the genetically corrected cells expressed normal β-globin transcripts. Our studies provide the most efficient and safe approach for the genetic correction of the β-41/42 (TCTT) deletion in iPSCs for further potential cell therapy of β-thal, which represents a potential therapeutic avenue for the gene correction of MGD-associated mutants in patient-specific iPSCs.
KW - CRISPR/Cas9
KW - induced pluripotent stem cells
KW - ssODNs
KW - β thalassemia
UR - http://www.scopus.com/inward/record.url?scp=85015672253&partnerID=8YFLogxK
U2 - 10.1016/j.omtn.2016.11.010
DO - 10.1016/j.omtn.2016.11.010
M3 - Article
AN - SCOPUS:85015672253
SN - 2162-2531
VL - 6
SP - 57
EP - 67
JO - Molecular Therapy Nucleic Acids
JF - Molecular Therapy Nucleic Acids
ER -