Therapeutic gene correction of HBB frameshift CD41-42 (-TCTT) deletion in human hematopoietic stem cells

作者：Qianyi Liu, Xinyu Li, Hui Xu, Ying Luo, Lin Cheng, Junbin Liang, Yuelin He, Haiying Liu, Jian‐Pei Fang, Junjiu Huang · 发表于：Advanced Biotechnology · 年份：2025 · DOI：10.1007/s44307-024-00053-5 · 被引用次数：5 · 研究领域：CRISPR and Genetic Engineering、Virus-based gene therapy research、Pluripotent Stem Cells Research

Β-thalassemia is one of the global health burdens. The CD41-42 (-TCTT) mutation at HBB is the most prevalent pathogenic mutation of β-thalassemia in both China and Southeast Asia. Previous studies focused on repairing the HBB CD41-42 (-TCTT) mutation in β-thalassemia patient-specific induced pluripotent stem cells, which were subsequently differentiated into hematopoietic stem and progenitor cells (HSPCs) for transplantation. In this study, we directly applied the CRISPR/Cas9-based gene editing therapy to correct the HBB CD41-42 (-TCTT) mutation in patient-derived HSPCs. The effective editing induced by Cas9:sgRNA ribonucleoprotein and single-stranded oligodeoxynucleotides (ssODNs) was confirmed in HUDEP-2 cell lines harboring the HBB CD41-42 (-TCTT) mutation. Further correction of heterozygote and homozygote HBB CD41-42 (-TCTT) mutations in patient-derived HSPCs resulted in a 13.4-40.8% increase in the proportion of HBB-expressing (HBB +) cells following erythroid differentiation in vitro. At 16 weeks post-xenotransplantation of the edited HSPCs into coisogenic immunodeficient mice, the reparation efficiency in engrafted bone marrow was 17.21% ± 3.66%. Multiparameter flow cytometric analysis of the engrafted bone marrow showed an increase in the percentage of HBB + cells without impairing the ability of engraftment, self-renewal, and multilineage hematopoietic repopulation of HSPCs. For the safety evaluation, 103 potential off-target sites were predicted by SITE-seq and CRIS...