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

• Published in: Molecular therapy. Nucleic acids Vol. 6; pp. 57 - 67
• Main Authors: Liu, Yali, Yang, Yi, Kang, Xiangjin, Lin, Bin, Yu, Qian, Song, Bing, Gao, Ge, Chen, Yaoyong, Sun, Xiaofang, Li, Xiaoping, Bu, Lei, Fan, Yong
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 17.03.2017; Elsevier Limited; American Society of Gene & Cell Therapy; Elsevier
• Subjects: Anemia; Cell differentiation; Chelation therapy; Clonal deletion; CRISPR; CRISPR/Cas9; Deoxyribonucleic acid; DNA; DNA fingerprints; Drug resistance; Efficiency; Erythroblasts; Gene deletion; Gene therapy; Genomes; Hematological diseases; Hematopoietic stem cells; Hemoglobin; induced pluripotent stem cells; Mutagenesis; Mutation; Nuclease; Oligonucleotides; Original; Patients; Pluripotency; Progenitor cells; ssODNs; Stem cells; Thalassemia; Therapeutic applications; Transplants & implants; β thalassemia; β thalassemia; induced pluripotent stem cells; CRISPR/Cas9; ssODNs
• ISSN: 2162-2531; 2162-2531
• DOI: 10.1016/j.omtn.2016.11.010

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.