Wongborisuth Chokdee, Innachai Pawarit, Saisawang Chonticha, Tubsuwan Alisa, Jearawiriyapaisarn Natee, Kaewprommal Pavita, Piriyapongsa Jittima, Chiangjong Wararat, Anurathapan Usanarat, Songdej Duantida, Tangprasittipap Amornrat, Hongeng Suradej
Offices of Health Science Research, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, 10400, Thailand.
Center for Advanced Therapeutics, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakhon Pathom, 73170, Thailand.
Sci Rep. 2025 Jul 15;15(1):25580. doi: 10.1038/s41598-025-10791-8.
CRISPR/Cas9 genome editing has emerged as a promising treatment for genetic diseases like β-thalassemia. Editing γ-globin promoters to disrupt ZBTB7A/LRF or BCL11A binding sites has shown potential for reactivating fetal hemoglobin and treating sickle cell disease. However, its application to β-thalassemia/HbE disease remains unclear. This study utilized CRISPR/Cas9 to disrupt these sites in mobilized CD34 + hematopoietic stem /progenitor cells from healthy donors and β-thalassemia/HbE patients. The editing efficiency for the BCL11A site (75-92%) was higher than for the ZBTB7A/LRF site (57-60%). Both disruptions similarly increased fetal hemoglobin production in healthy donors (BCL11A 26.2 ± 1.4%, ZBTB7A/LRF 27.9 ± 1.5%) and β-thalassemia/HbE cells (BCL11A 62.7 ± 0.9%, ZBTB7A/LRF 64.0 ± 1.6%). Off-target effects were absent in BCL11A-edited cells but observed at low frequencies in ZBTB7A/LRF-edited cells. Neither disruption significantly affected erythroid differentiation. These findings highlight the comparable contributions of ZBTB7A/LRF and BCL11A binding sites to γ-globin reactivation. CRISPR/Cas9 editing of either site may offer a potential therapeutic strategy for β-thalassemia/HbE disease.
CRISPR/Cas9基因编辑已成为治疗诸如β地中海贫血等遗传性疾病的一种有前景的方法。编辑γ珠蛋白启动子以破坏ZBTB7A/LRF或BCL11A结合位点已显示出重新激活胎儿血红蛋白并治疗镰状细胞病的潜力。然而，其在β地中海贫血/HbE疾病中的应用仍不明确。本研究利用CRISPR/Cas9破坏健康供体以及β地中海贫血/HbE患者动员的CD34 +造血干/祖细胞中的这些位点。BCL11A位点的编辑效率（75 - 92%）高于ZBTB7A/LRF位点（57 - 60%）。两种破坏在健康供体（BCL11A 26.2±1.4%，ZBTB7A/LRF 27.9±1.5%）和β地中海贫血/HbE细胞（BCL11A 62.7±0.9%，ZBTB7A/LRF 64.0±1.6%）中同样增加了胎儿血红蛋白的产生。在BCL11A编辑的细胞中未发现脱靶效应，但在ZBTB7A/LRF编辑的细胞中观察到低频脱靶效应。两种破坏均未显著影响红系分化。这些发现突出了ZBTB7A/LRF和BCL11A结合位点对γ珠蛋白重新激活的类似贡献。对任一位点进行CRISPR/Cas9编辑可能为β地中海贫血/HbE疾病提供一种潜在的治疗策略。
