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

ABSTRACT: Monogenic disorders (MGDs), which are caused by single gene mutations, have a serious effect on human health. Among these, ?-thalassemia (?-thal) represents one of the most common hereditary hematological diseases caused by mutations in the human hemoglobin ? (HBB) gene. The technologies of induced pluripotent stem cells (iPSCs) and genetic correction provide insights into the treatments for MGDs, including ?-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 ?-41/42 (TCTT) deletion mutation in ? 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 ?-globin transcripts. Our studies provide the most efficient and safe approach for the genetic correction of the ?-41/42 (TCTT) deletion in iPSCs for further potential cell therapy of ?-thal, which represents a potential therapeutic avenue for the gene correction of MGD-associated mutants in patient-specific iPSCs.

PROVIDER: S-EPMC5363452 | BioStudies |

REPOSITORIES: biostudies

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