Heterogeneity of HbF levels in β-thalassemia/HbE disease has been reported to be associated with variations in clinical manifestations of the disease, and several genetic-modifying factors beyond the β-globin gene cluster have been identified as HbF regulators. Down-regulation or heterozygous mutations of Krüppel-like factor 1 (KLF1) is associated with elevated HbF levels in non-thalassemia subjects. This study confirms that experimental down-regulation of KLF1 in β-thalassemia/HbE-derived erythroblasts significantly increases HbF production (up to 52.3 ± 2.4%), albeit with slightly delayed erythroid terminal differentiation. KLF1 exome sequencing of 130 Thai β-thalassemia/HbE patients without co-inheritance of α-thalassemia found six patients with KLF1 heterozygous mutations including rs2072596 (p.F182L; n = 5) and rs745347362 (p.P284L; n = 1) missense mutations. However, while these patients had high HbF levels (38.1 ± 7.5%), they were all associated with a severe clinical phenotype. These results suggest that while reduction of KLF1 expression in β-thalassemia/HbE erythroblasts can increase HbF levels, it is not sufficient to alleviate the clinical phenotype.
LRF/ZBTB7A is a transcription factor that has been recently identified as a new key regulator of fetal hemoglobin (HbF; α2γ2) production in erythroid cells. Reduction of LRF/ZBTB7A expression led to increases in levels of HbF in human CD34+ hematopoietic stem and progenitor cell (HSPC)-derived erythroblast and in human immortalized erythroid line (HUDEP-2). Since reactivation of γ-globin gene is associated with the improvement of clinical manifestations of β-hemoglobinopathy patients, decrement in LRF/ZBTB7A expression might be a substantial interest as a novel target for gene therapy in β-thalassemia. In this study, we investigated the effects of LRF/ZBTB7A downregulation in erythroid cells derived from β-thalassemia/HbE patients in order to evaluate its therapeutic potential. The hematopoietic CD34+ progenitor cells were collected from 3 patients and 3 healthy normal individuals' peripheral blood and subjected for in vitro erythroblast culture. The cells were transduced with lentivirus carrying LRF/ZBTB7A specific shRNA, and used untransduced cells and non-targeted control shRNA (shNTC) as experimental controls. The LRF/ZBTB7A shRNA reduced LRF/ZBTB7A transcript and protein to nearly undetectable levels. Interestingly, downregulation of LRF/ZBTB7A increased expression of γ-globin, ε-globin and ζ-globin in both adult normal and β-thalassemia/HbE derived cells, whereas α-globin, β-globin and δ-globin expression were decreased. As previously reported, we found that the LRF/ZBTB7A knockdown produced a robust increase in HbF levels in both normal (43.3±9.0% vs. 5.9±2.1% in shNTC) and β-thalassemia/HbE erythroblasts (78.1±3.5% vs. 26.3±3.9% in shNTC). Noteworthy, the delay of erythroid differentiation was observed in the LRF/ZBTB7A knockdown cells of both derived from β-thalassemia/HbE patients and normal control, suggesting an additional role of LRF/ZBTB7A in regulating erythroid maturation. These data support the manipulation of LRF/ZBTB7A as one of the most interesting gene therapy candidates for treating the β-thalassemia, but the effect on erythroid cell maturation is needed to be concerned and required further investigation.
Disclosures
No relevant conflicts of interest to declare.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.