One-Step Biallelic and Scarless Correction of a β-Thalassemia Mutation in Patient-Specific iPSCs without Drug Selection

Yali Liu; Yi Yang; Xiangjin Kang; Bin Lin; Qian Yu; Bing Song; Ge Gao; Yaoyong Chen; Xiaofang Sun; Xiaoping Li; Lei Bu; Yong Fan

doi:10.1016/j.omtn.2016.11.010

One-Step Biallelic and Scarless Correction of a β-Thalassemia Mutation in Patient-Specific iPSCs without Drug Selection

Mol Ther Nucleic Acids. 2017 Mar 17:6:57-67. doi: 10.1016/j.omtn.2016.11.010. Epub 2016 Dec 10.

Authors

Yali Liu¹, Yi Yang¹, Xiangjin Kang¹, Bin Lin², Qian Yu¹, Bing Song¹, Ge Gao³, Yaoyong Chen¹, Xiaofang Sun¹, Xiaoping Li⁴, Lei Bu⁵, Yong Fan⁶

Affiliations

¹ Key Laboratory for Major Obstetric Diseases of Guangdong Province, Key Laboratory of Reproduction and Genetics of Guangdong Higher Education Institutes, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510150, China.
² Leon H. Charney Division of Cardiology, New York University School of Medicine, 522 First Avenue, New York, NY 10016, USA.
³ Key Laboratory of Regenerative Biology of the Chinese Academy of Sciences and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
⁴ Key Laboratory of Regenerative Biology of the Chinese Academy of Sciences and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China. Electronic address: li_xiaoping@gibh.ac.cn.
⁵ Leon H. Charney Division of Cardiology, New York University School of Medicine, 522 First Avenue, New York, NY 10016, USA. Electronic address: lei.bu@med.nyu.edu.
⁶ Key Laboratory for Major Obstetric Diseases of Guangdong Province, Key Laboratory of Reproduction and Genetics of Guangdong Higher Education Institutes, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510150, China. Electronic address: yongfan011@gzhmu.edu.cn.

Abstract

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.

Keywords: CRISPR/Cas9; induced pluripotent stem cells; ssODNs; β thalassemia.