AUTHOR=Yang Yinghong , He Lina , Xie Yingjun , Zhu Lifen , Wu Jianfeng , Fan Yong , Yang Yi , Sun Xiaofang TITLE=In situ correction of various β-thalassemia mutations in human hematopoietic stem cells JOURNAL=Frontiers in Cell and Developmental Biology VOLUME=11 YEAR=2024 URL=https://www.frontiersin.org/journals/cell-and-developmental-biology/articles/10.3389/fcell.2023.1276890 DOI=10.3389/fcell.2023.1276890 ISSN=2296-634X ABSTRACT=

β-thalassemia (β-thal) is the most common monogenic disorder caused by various mutations in the human hemoglobin β (HBB) gene and affecting millions of people worldwide. Electroporation of Cas9 and single-guide RNA (sgRNA)–ribonucleoprotein (RNP) complex-mediated gene targeting in patient-derived hematopoietic stem cells (HSCs), followed by autologous transplantation, holds the promise to cure patients lacking a compatible bone marrow donor. In this study, a universal gene correction method was devised to achieve in situ correction of most types of HBB mutations by using validated CRISPR/sgRNA–RNP complexes and recombinant adeno-associated viral 6 (rAAV6) donor-mediated homology-directed repair (HDR) in HSCs. The gene-edited HSCs exhibited multi-lineage formation abilities, and the expression of β-globin transcripts was restored in differentiated erythroid cells. The method was applied to efficiently correct different mutations in β-thal patient-derived HSCs, and the edited HSCs retained the ability to engraft into the bone marrow of immunodeficient NOD-scid-IL2Rg−/− (NSI) mice. This study provides an efficient and safe approach for targeting HSCs by HDR at the HBB locus, which provides a potential therapeutic approach for treating other types of monogenic diseases in patient-specific HSCs.