A one-base therapeutic insertion in the HBG2 distal promoter reactivates γ-globin expression

ABSTRACT: The reactivation of developmental silenced g-globin genes (HBG1/2) has shown promise as a therapeutic strategy for improving symptoms of b-hemoglobinopathies. Currently, the focus of therapeutic targets is primarily on the major fetal hemoglobin suppressors, such as BCL11A and ZBTB7A and of their binding sites on the proximal HBG promoter. However, the role of the distal HBG promoter in regulating gene expression remains to be explored. Here, we discovered an insertion of nucleotide A (insA) between -1368 and -1369 bp upstream of the TSS in HBG2 resulting in remarkable increase in γ-globin expression in HUDEP-2 cells. We also observed elevated γ-globin expression in human CD34+ erythroid progenitor cells from healthy individuals and those with b-thalassemia when introducing insA mutation. Similarly, engrafted NCG-Kit-V831M mice showed increased γ-globin expression. Importantly, neither did insA have any off-target effects nor did it affect the maturation of erythroid cells. Furthermore, we found that the insA mutation created a binding site for the transcription activator FOXO3, which can reactivate g-globin. Additionally, introducing insA specifically and significantly demethylated the -162 CpG site on HBG promoter by reducing the enrichment of DNA methyltransferase 3A (DNMT3A). At the same time, it activated histone modifications and RNA polymerase II (Pol II) in both distal and proximal HBG promoter and inhibited the binding of BCL11A and ZBTB7A on -115 and -200 sites on the HBG promoter respectively. Overall, our study suggests that introducing insA mutation leads to significantly boosted fetal globin levels and uncovers new safe therapeutic target or strategy for β-hemoglobinopathies.

ORGANISM(S): Homo sapiens

PROVIDER: GSE283117 | GEO | 2025/04/02

REPOSITORIES: GEO