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=Volume 11 - 2023

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) RNP complexes 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 rAAV6-donor mediated homology directed repair (HDR) in HSCs. The gene edited HSCs exhibited multilineage 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 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.